WO2013025052A2 - Bioactive carbon nanotube composite functionalized with β-sheet polypeptide block copolymer, and preparation method thereof - Google Patents

Bioactive carbon nanotube composite functionalized with β-sheet polypeptide block copolymer, and preparation method thereof Download PDF

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WO2013025052A2
WO2013025052A2 PCT/KR2012/006502 KR2012006502W WO2013025052A2 WO 2013025052 A2 WO2013025052 A2 WO 2013025052A2 KR 2012006502 W KR2012006502 W KR 2012006502W WO 2013025052 A2 WO2013025052 A2 WO 2013025052A2
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sheet
polypeptide block
block copolymer
complex
peptide
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PCT/KR2012/006502
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French (fr)
Korean (ko)
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WO2013025052A3 (en
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임용범
정우진
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연세대학교 산학협력단
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Priority to US14/239,426 priority Critical patent/US9364550B2/en
Priority to CN201280045387.1A priority patent/CN103827187B/en
Publication of WO2013025052A2 publication Critical patent/WO2013025052A2/en
Publication of WO2013025052A3 publication Critical patent/WO2013025052A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/15—Nano-sized carbon materials
    • C01B32/158—Carbon nanotubes
    • C01B32/168—After-treatment
    • C01B32/174—Derivatisation; Solubilisation; Dispersion in solvents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/02—Elements
    • C08K3/04—Carbon
    • C08K3/041—Carbon nanotubes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K9/00—Use of pretreated ingredients
    • C08K9/04—Ingredients treated with organic substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to a bioactive carbon nanotube complex functionalized with a ⁇ -sheet polypeptide block copolymer, and more particularly, to water dispersible carbon nanotubes using an amphipathic ⁇ -sheet polypeptide block copolymer having bioactivity. Functionalized as an excellent bioactive complex, and utilized as a composition for intracellular delivery of biosensors and biologically active substances.
  • Carbon nanotubes are nanomaterials with a wide range of electrical, optical, mechanical and biological applications. To use CNTs as biomaterials, several physical, chemical and biological characteristics must be met. CNTs are essentially insoluble in aqueous solutions and heavily aggregated. Since the biological system is basically composed of water-soluble molecules and structures, the problem of solubilization in aqueous solution has to be solved for the biological use of CNTs. It is also important to functionalize the surface with bioactive molecules in order to allow CNTs to perform specific biological functions.
  • ⁇ -conjugation systems of CNTs can be preserved in non-covalent functionalization methods.
  • This method mainly uses amphiphilic molecules, where the hydrophobic portion of the amphiphilic molecule surrounds the walls of the CNT and the hydrophilic portion interacts with the aqueous solution.
  • Low molecular weight molecules or surfactants, polymers, carbohydrates and the like have been used for non-covalent functionalization and solubilization of CNTs.
  • Korean Patent Publication (Publication No. 10-2004-0075620) describes the functionalization of carbon nanotubes in which carbon nanotubes / nucleic acid conjugates are formed through non-covalent bonds in a solid state
  • a Korean registered patent ( Registration No. 10-0682381) describes a carbon nanotube derivative in which a carbon nanotube and an egg white protein are non-covalently bound.
  • the first object of the present invention is to provide a carbon nanotube complex functionalized using a bioactive ⁇ -sheet polypeptide block copolymer formed by conjugating a bioactive and hydrophilic peptide portion with a ⁇ -sheet portion.
  • the second problem to be solved by the present invention is to provide a composition for intracellular delivery of a biosensor and a biologically active substance using a carbon nanotube complex functionalized using the ⁇ -sheet polypeptide block copolymer.
  • the third object of the present invention is to provide a method for producing a carbon nanotube complex functionalized using the ⁇ -sheet polypeptide block copolymer.
  • the present invention to achieve the first object,
  • the ⁇ -sheet polypeptide block copolymer consists of a ⁇ -sheet polypeptide block and a bioactive polypeptide block,
  • the ⁇ -sheet polypeptide block has a structure in which non-polar amino acids and polar amino acids are alternately repeated, or 50-100% of non-polar amino acids,
  • the bioactive polypeptide block is composed of polar amino acids 50-100% of the amino acids constituting the bioactive polypeptide block,
  • the ⁇ -sheet polypeptide block is non-covalently bonded to the surface of the carbon nanotubes
  • the bioactive polypeptide block is hydrophilic and provides a complex of ⁇ -sheet polypeptide block copolymer and carbon nanotube, which is exposed to the outside of the complex.
  • the non-polar amino acid is phenylalanine (phenylalanine), alanine (alanine), valine (valine), isoleucine (isoleucine), leucine, methionine (methionine), tyrosine (tyrosine) and tryptophan ( tryptophan).
  • the polar amino acids are each independently lysine (glycine), glycine (glycine), arginine (arginine), proline (proline), glutamine (glutamine), serine, histidine (histidine) , Aspartic acid (glucomic acid), glutamic acid (glutamic acid), threonine (threonine), asparagine (aspargine), cysteine (cysteine) and selenocysteine (selenocysteine).
  • the non-polar amino acid of the ⁇ -sheet polypeptide block is phenylalanine or tryptophan, and the polar amino acid may be lysine, glutamic acid or glycine.
  • the ⁇ -sheet polypeptide block forms ⁇ -sheet hydrogen bonds between peptide strands to form self-assembly, and the phenylalanine or tryptophan stacks ⁇ - ⁇ with the surface of the carbon nanotubes. And non-covalent bonds through hydrophobic interactions.
  • the ⁇ -sheet polypeptide block copolymer may be a macrocyclic peptide having an N-terminus and a C-terminus bonded thereto.
  • the present invention to achieve the second object,
  • a ligand or a receptor capable of reacting with a target biomaterial is attached to the ⁇ -sheet polypeptide block copolymer and the carbon nanotube complex, and the ⁇ -sheet polypeptide block copolymer and the carbon nanotube complex. It provides a biosensor.
  • the ligand and receptor may be an enzyme substrate, a ligand, an amino acid, a peptide, a protein, an enzyme, a lipid, a cofactor, a carbohydrate, and a combination of the two.
  • the present invention also provides a composition for intracellular delivery of a biologically active substance comprising a complex of the ⁇ -sheet polypeptide block copolymer and a carbon nanotube as an active ingredient.
  • the present invention to achieve the third object,
  • the dispersion may further comprise a salt.
  • the suspension solvent of step (a) is tetrahydrofuran
  • the suspension solvent may be removed by a centrifugation method.
  • the concentration of the salt in the dispersion is 1 mM or more
  • the dispersion may be prepared using an ultrasonic method.
  • the present invention is the functionalization of carbon nanotubes using an amphipathic ⁇ -sheet polypeptide block copolymer having a bioactivity, and the peptide / carbon nanotube complexes prepared according to the present invention are excellent in water dispersibility and have bioactivity to stimulate responsiveness. It can be used as a biomaterial or in the manufacture of CNT based electronic biosensor devices. In addition, it can be used as a composition for intracellular delivery of a biologically active substance, and may be useful for the design and development of inhibitors for diseases caused by abnormal folding of proteins by applying the interaction between ⁇ -sheet peptides and carbon-based hydrophobic substances. It is expected to be.
  • FIG. 1 is a conceptual diagram showing a reaction route of various combinations / self-assembly of ⁇ -sheet polypeptide block copolymers and single-walled carbon nanotubes (SWCNTs) according to the present invention, wherein a part (GRKKRRQRRRPPQGSGG) of the peptide is a bioactive part, Part b (FKFEFKFEFKFE) is the ⁇ -sheet self-assembled part.
  • GRKKRRQRRRPPQGSGG a part of the peptide
  • Part b FKFEFKFEFKFE
  • (b) is a graph showing the hydration radius ( R H ) distribution of the solubilized peptide / SWCNT complex, as measured by dynamic light scattering (DLS),
  • (c) is an image confirming the effect of ⁇ -sheet polypeptide block copolymer concentration on the solubilization of SWCNTs from the left at peptide concentrations 1.5, 5 and 12.5 mM. (The amount of SWCNT is 5 mg)
  • (a) is an image showing the vortexed peptide sample in pure water (left) or 50 mM NaCl (right).
  • (b) is an image showing the sonicated peptide sample in pure water (left) or 50 mM NaCl (right).
  • (c) is a graph showing the CD spectrum, a (thick solid line, 5 mM peptide + 60 mM NaCl), b (dotted line, 5 mM peptide + 20 mM NaCl), c (double-stranded line, 5 mM peptide + 60 mM NaCl) + 5 mg SWCNT), d (thin solid, 5 mM peptide + 20 mM NaCl + 5 mg SWCNT).
  • (d) is a graph showing the fluorescence emission spectra of the peptide single layer (b, dotted line) and the peptide / SWCNT complex (a, solid line).
  • (e) and (f) are TEM images of solubilized peptide / SWCNT (50 mM / 5 mg) complexes in pure water (e) and in the presence of 20 mM NaCl (f), respectively.
  • (a) is an image showing the intracellular distribution of the peptide / SWCNT complex
  • (a) is the CD spectrum measured at various salt concentrations.
  • (b) is a graph showing the ellipticity of molecules according to the ⁇ -sheet content in the ⁇ -sheet polypeptide block copolymer measured at 215 nm.
  • Figure 6 confirms the effect of ionic strength on the ⁇ -sheet formation of the polypeptide block copolymer
  • (a) is CD spectrum measured at various salt (0-120 mM) concentrations.
  • (b) is a graph showing the ellipticity of molecules according to the ⁇ -sheet content in the ⁇ -sheet polypeptide block copolymer measured at 215 nm.
  • Fig. 7 shows the structure of the cyclic ⁇ -sheet polypeptide block copolymer (peptides 1 to 4 of Synthesis Example 2) according to the present invention.
  • FIG. 11 is an image confirming SWCNT solubilization of a cyclic peptide according to the present invention to explore the availability of tryptophan residues in addition to phenylalanine residues as ⁇ -sheet forming portions of polypeptide copolymers for solubilization of SWCNTs.
  • FIG. 12 is an image showing that SWCNTs can be solubilized by varying the number and arrangement of ⁇ -sheet forming residues, particularly tryptophan residues, of the ⁇ -sheet polypeptide portion.
  • Figure 13 is a conceptual diagram showing a cyclization process in the synthesis of cyclic ⁇ -sheet polypeptide block copolymer according to the present invention.
  • Peptides minimized forms of proteins
  • Peptides can be developed into protein-like artificial nanomaterials by forming controllable nanostructures through proper design and self-assembly.
  • self-assembled ⁇ -sheet peptides can be functionalized into bioactive nanostructures by conjugating bioactive and hydrophilic peptide moieties with ⁇ -sheet moieties to form bioactive ⁇ -sheet polypeptide block copolymers. This enables the production of various forms of controllable and biologically useful ⁇ -sheet peptide nanostructures.
  • ⁇ -sheet polypeptide block copolymers can self-assemble themselves to form nanostructures.
  • controlling the self-assembly of the ⁇ -sheet polypeptide block copolymer enables co-assembly with carbon nanotubes instead of self-assembly.
  • the present invention provides a technique of co-self-assembling ⁇ -sheet polypeptide block copolymer and carbon nanotube to make ⁇ -sheet polypeptide block copolymer / carbon nanotube complex.
  • the present invention provides a self-assembled bioactive carbon nanotube complex by combining a bioactive ⁇ -sheet polypeptide block copolymer and a CNT.
  • a plurality of ⁇ -sheet polypeptide block copolymers in which a bioactive polypeptide and a ⁇ -sheet polypeptide are copolymerized surround all or a portion of the carbon nanotube surface by non-covalent bonds, and the ⁇ -sheet polypeptide portion is bonded to the carbon nanotube surface.
  • the hydrophilic polypeptide portion is charged and exposed to the outer surface layer, and the bioactive polypeptide is composed of a plurality of repeating one or more amino acids selected from lysine, glycine, arginine, proline, glutamine and serine, and the ⁇
  • the -sheet polypeptide provides a ⁇ -sheet polypeptide block copolymer-carbon nanotube water-soluble complex, characterized in that a plurality of one or more amino acids selected from phenylalanine, tryptophan, lysine, glutamic acid and glycine are repeated in plurality.
  • the ⁇ -sheet polypeptide block copolymer may be a linear peptide or a macrocyclic peptide having an N-terminus and a C-terminus bonded thereto.
  • two competing forces i.e., the attraction between the ⁇ -sheet portion and the attraction between the ⁇ -sheet portion and the CNT
  • the former dominates the peptide participates in the formation of ⁇ -sheet nanostructures but the CNTs remain insoluble in the aqueous solution.
  • the latter prevails water-soluble / bioactive CNTs that are non-covalently functionalized by bioactive ⁇ -sheet polypeptide block copolymers can be obtained.
  • the balance between the competitive forces is elucidated in detail, it may be possible to control and prevent aggregation of ⁇ -amyloid and related proteins in diseases caused by abnormal folding of proteins.
  • the ⁇ -sheet polypeptides are self-assembled by forming ⁇ -sheet hydrogen bonds between peptide strands, and the phenylalanine or tryptophan residues of the ⁇ -sheet polypeptides interact with each other through a ⁇ - ⁇ stacking interaction. It is characterized by combining with.
  • the ⁇ -sheet polypeptide block copolymer consists of two parts having very different characteristics in polarity and aggregation tendency.
  • the bioactive moiety (eg, part a of FIG. 1 below) is based on Tat, a cell permeable peptide. This part contains a large number of lysine and arginine, so it is highly charged and polarized in aqueous solution.
  • the self-assembled moiety has a repeating structure of nonpolar amino acids (phenylalanine or tryptophan) and polar amino acids (lysine and glutamic acid) and promotes ⁇ -sheet hydrogen bond formation between peptide strands.
  • Polypeptide block copolymers having this structure can form ⁇ -sheet nanoribbon structures only under sufficiently high ionic strength because of the large volume of the hydrophilic moiety and the large number of positive charges (Path 1 in FIG. 1 below). If no salt is present or the ionic strength is low, the polypeptide block copolymer is mainly in the form of a random coil.
  • the present invention provides a biosensor comprising attaching a ligand or a receptor reacting with a target biomaterial to the ⁇ -sheet polypeptide block copolymer-carbon nanotube-soluble complex.
  • the target biomaterial or organic compound is a substance capable of serving as a target to be detected by reacting with a ligand or receptor, preferably a protein, nucleic acid, antibody, enzyme, carbohydrate, lipid or other biomolecule, and more.
  • a ligand or receptor preferably a protein, nucleic acid, antibody, enzyme, carbohydrate, lipid or other biomolecule, and more.
  • proteins, nucleic acids, and carbohydrates associated with the disease are preferred.
  • the ligand or receptor may be characterized in that the enzyme substrate, ligand, amino acids, peptides, proteins, nucleic acids, lipids, cofactors or carbohydrates.
  • the present invention also provides a composition for intracellular delivery of a biologically active substance comprising a ⁇ -sheet polypeptide block copolymer-carbon nanotube water-soluble complex as an active ingredient.
  • composition of the present invention allows the biologically active substance to act directly in the cell through the cell membrane, which has not been able to easily pass through. Therefore, the composition of the present invention can be a breakthrough in the development of drug delivery system (drug delivery system).
  • composition of the present invention comprises 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition.
  • composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.
  • composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as needed.
  • other conventional additives such as buffers and bacteriostatic agents can be added.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs.
  • Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used.
  • it may be preferably formulated according to each disease or component by an appropriate method in the art.
  • composition comprising the fusion as an active ingredient is intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, nasal, mucosal, mucosal, It can be delivered in vivo by infusion, such as by inhalation and oral. Dosage varies depending on the subject's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of disease.
  • the daily dosage is about 0.1 to 100 mg / kg for the compound, preferably 0.5 to 10 mg / kg, and more preferably administered once to several times a day.
  • the present invention comprises the steps of (a) suspending carbon nanotubes in tetrahydrofuran and centrifuging to evaporate tetrahydrofuran to obtain pretreated carbon nanotubes, and (b) the pretreated carbon nanotubes, 5-200. It provides a method for producing a ⁇ -sheet polypeptide block copolymer-carbon nanotube water-soluble complex comprising the step of mixing and sonicating the ⁇ M ⁇ -sheet polypeptide block copolymer aqueous solution and 10-150 mM sodium chloride aqueous solution.
  • the ⁇ -sheet polypeptide block copolymer is a block copolymer of a bioactive polypeptide and a ⁇ -sheet polypeptide, and the bioactive polypeptide is one or more amino acids selected from lysine, glycine, arginine, proline, glutamine and serine in a plurality of repetitions.
  • the ⁇ -sheet polypeptide is characterized in that a plurality of repeating one or more amino acids selected from phenylalanine, tryptophan, lysine, glutamic acid and glycine.
  • Fmoc-amino acids were purchased from Novabiochem (Germany) and used with Fmoc-21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid (Fmoc-NH-PEG5-COOH).
  • N- (Fmoc-8-amino-3,6-dioxaoctyl) succinamic acid Fmoc-PEG2-Suc-OH or Fmoc-Ebes-OH
  • Single-walled carbon nanotubes (ASP-100F, "refined” grades) prepared by the arc discharge method were purchased from Hanwha Nanotech (Korea).
  • Tissue culture reagents were purchased from Invitrogen (USA).
  • the molecular weight was determined by MALDI-TOF mass spectrometry. HPLC analysis showed that the purity of the peptide was at least 95%.
  • CD spectra were measured using a Chirascan TM circular dichroism spectrometer equipped with a Peltier thermostat (Applied Photophysics., Ltd). Spectra were measured from 250 nm to 190 nm using cuvettes of 2 mm path length. The average value was recorded by measuring 5 times repeatedly. The mole ellipticity was calculated for each amino acid residue. Prior to the measurement, 5 ⁇ M peptide solution with or without SWCNTs was treated with 20 mM or 60 mM NaCl for 12 hours at room temperature.
  • DLS experiments were performed at room temperature using an ALV / CGS-3 Compact Goniometer System equipped with a 632.8 nm He-Ne laser.
  • an ALV / SO-SIPD / DUAL detector with an optical fiber, an EMI PM-28B power supply, and an ALV / PM-PD preamplifier / identifier were used.
  • the signal analyzer was an ALV-5000 / E / WIN multiple tower digital correlator with 288 exponentially arranged channels. The scattering angle was 90 degrees.
  • the size distribution was determined by the forced adjustment method.
  • HeLa cells (4 ⁇ 10 5 ) were dispensed into an 8-well Lab-tek II chamber cover glass system (Nunc) and 10% FBS It was incubated overnight at 37 °C using DMEM containing. Cells were washed with DPBS and then treated with peptide / SWCNT complex for 20 minutes. The sample solution was then removed and the cells were further incubated for 90 minutes in DMEM. Prior to imaging, LysoTracker Red DND-99 (Invitrogen) at 50 nM concentration was added for 5 minutes. Confocal images were taken using a Nikon Eclipse TE2000-U inverted microscope equipped with argon (488 nm) and helium-neon (543 nm) lasers.
  • this peptide capable of forming ⁇ -sheets could bind to SWCNTs solubilizing SWCNTs in pure water.
  • SWCNT prepared by the arc discharge method was suspended in tetrahydrofuran (THF) and the same amount (5 mg) of SWCNT was added to the microcentrifuge tube. After THF evaporated, the polypeptide block copolymer solution (0.3 mL) was added and the mixture was sonicated for 15 minutes at room temperature.
  • THF tetrahydrofuran
  • zeta potential ( ⁇ ) measurements showed that the peptide / SWCNT complex had a large value (+ 58 ⁇ 3 mV), from which the positively charged hydrophilic portion of the outer layer of the peptide / SWCNT complex was exposed. It can be seen that a surface having a positive charge is formed.
  • salt further enhances the hydrophobic interaction between phenylalanine and SWCNT and blocks the nonspecific, electrical interaction between peptides, contributing to the effective solubilization and aggregation inhibition of SWCNTs.
  • bioactive CNTs functionalized with ⁇ -sheet polypeptide block copolymers by combining pathways 2 and 3 of FIG. It can manufacture.
  • amyloid fibers in diseases caused by abnormal folding of proteins. That is, allotropes of carbon (eg, CNTs and fullerenes) can inhibit the formation of amyloid, considering that it is difficult to decompose the already assembled ⁇ -sheet nanoribbons (paths 4 and 5).
  • the main target of inhibitor development will be intermediate assemblies such as fibrillar species produced in the early stages of amyloid fiber formation.
  • the complex is modified with Tat, a cell permeable peptide, so it enters the cell easily. Tat passes through cell membranes and nuclear membranes.
  • peptides labeled with fluorescein and unlabeled peptides were mixed in a molar ratio of 1:50 and the peptide mixture was used to functionalize SWCNTs.
  • Synthesis Peptides were synthesized using Tribute TM Peptide Synthesizer (Protein Technologies, Inc) according to standard Fmoc protocol on Rink Amide MBHA Resin LL (Novabiochem), except for Cysteine with methoxytrytil (Mmt) protecting group during synthesis. Standard amino acid protecting groups were used for the remaining amino acids.
  • Synthesis of the synthesized ⁇ -sheet polypeptide block copolymers (following 1 to 4 peptide) is as follows.
  • the block copolymers are macrocyclic peptides in which an N-terminal portion and a C-terminal portion are bonded through a cyclization reaction shown in FIG. 13, and the structure thereof is shown in FIG. 7 (blue). Bioactive peptide, and the part marked red is the ⁇ -sheet self-assembled part).
  • the resin was then washed with NMP and acetonitrile and dried under vacuum.
  • the resulting peptide was purified by reverse phase HPLC (water-acetonitrile, 0.1% TFA).
  • the molecular weight was determined by MALDI-TOF mass spectrometry. HPLC analysis showed that the purity of the peptide was 95% or more.
  • Peptide No. 1 in water acetonitrile (1: 1) using the tryptophan molar extinction coefficient at 280 nm, and peptide No. 2 using the phenylalanine molar absorption coefficient (195 m ⁇ 1 cm ⁇ 1 ). The concentration was measured spectroscopically at 257.5 nm.
  • CD spectra were measured using a Chirascan TM circular dichroism spectrometer equipped with a Peltier thermostat (Applied Photophysics., Ltd). Spectra were measured from 260 nm to 200 nm using cuvettes of 2 mm path length. The average value was recorded by measuring 5 times repeatedly. The mole ellipticity was calculated for each amino acid residue. Prior to the measurement, 50 ⁇ M peptide solution with or without SWCNTs was treated with 150 mM KF at room temperature and another 50 ⁇ M peptide solution without SWCNTs in distilled water for 24 hours each.
  • SWCNT prepared by the arc discharge method was suspended in tetrahydrofuran (THF) and the same amount (5 mg) of SWCNT was added to the microcentrifuge tube. After THF evaporated, the solution of No. 1 cyclic polypeptide block copolymer of Synthesis Example 2 (0.3 mL) was added, and the mixture was sonicated at room temperature for 15 minutes.
  • THF tetrahydrofuran
  • peptide 1 contained in a low concentration (20 mM) salt solution did not effectively solubilize SWCNT, whereas peptide 1 in a 150 mM salt concentration solution aided in sonication.
  • cyclic polypeptide copolymers like linear polypeptide copolymers, enhance the solubility of peptide / SWCNT complexes through hydrophobicity and ⁇ - ⁇ stacking interactions of phenylalanine residues and aqueous solutions of bioactive moieties at appropriate ionic strengths. It can be seen that.
  • CD spectra were measured to confirm peptide ⁇ -helix stabilization function of the prepared peptide / SWCNT complex.
  • the peptide without SWCNT did not show stabilization of ⁇ -helical structure in water and salt solution, but the CD spectrum of peptide / SWCNT complex in salt solution was observed at 208 nm and 222 nm.
  • the ⁇ -helical structure is effectively stabilized. From these results, SWCNTs strongly support the ⁇ -sheet-forming part of the cyclic polypeptide copolymer through hydrophobic and ⁇ - ⁇ stacking interactions, similar to the macromolecules in the body that support the ⁇ -helix structure. It can be seen that the structure is stabilized.
  • Cyclic peptide 2 was applied to SWCNT solubilization to explore the availability of tryptophan residues in addition to phenylalanine residues as ⁇ -sheet forming parts of the polypeptide copolymers for solubilization of SWCNTs.
  • the 40 mM aqueous KF salt solution did not suspend the SWCNT well (left image) while the peptide of 30 ⁇ M concentration contained in the 40 mM aqueous salt solution showed a tendency to solubilize the SWCNT with the help of sonication. It was confirmed (right image).
  • Bioactive ⁇ -sheet polypeptide block copolymers can self-assemble themselves and, if desired, also be used for functionalization of CNT-complexes. This is important for broadening the application of bioactive ⁇ -sheet polypeptide block copolymers.
  • Bioactive ⁇ -sheet polypeptide block copolymers / CNT complexes can be used as stimuli reactive biomaterials or in the fabrication of CNT based electronic biosensor devices.
  • understanding of the interaction between ⁇ -sheet peptides and carbon-based hydrophobic materials is expected to be useful in the design and development of inhibitors for diseases caused by abnormal folding of proteins.

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Abstract

The present invention relates to a bioactive carbon nanotube composite functionalized with a β-sheet polypeptide block copolymer which shows excellent water dispersion, and has biological activity so as to be used as a stimulus-reactive biomaterial or in the manufacture of CNT-based electronic biosensor devices. In addition, the bioactive carbon nanotube composite can be used as a composition for delivery of a biological active material into cells. Further, the application of the interaction between a β-sheet peptide and a carbon-based hydrophobic material is expected to be useful for designing and developing an inhibitor for diseases caused by the abnormal folding of a protein.

Description

베타-시트 폴리펩티드 블록 공중합체로 기능화된 생체활성 탄소나노튜브 복합체 및 그 제조방법Bioactive Carbon Nanotube Complexes Functionalized with Beta-Sheet Polypeptide Block Copolymers and Methods for Manufacturing the Same
본 발명은 β-시트 폴리펩티드 블록 공중합체로 기능화된 생체활성 탄소나노튜브 복합체에 관한 것으로서, 더욱 상세하게는 생체활성을 갖는 양친매성 β-시트 폴리펩티드 블록 공중합체를 이용하여 탄소나노튜브를 수분산성이 우수한 생체활성 복합체로 기능화하고, 이를 바이오 센서 및 생물학적 활성 물질의 세포내 전달용 조성물로 활용한 것이다.The present invention relates to a bioactive carbon nanotube complex functionalized with a β-sheet polypeptide block copolymer, and more particularly, to water dispersible carbon nanotubes using an amphipathic β-sheet polypeptide block copolymer having bioactivity. Functionalized as an excellent bioactive complex, and utilized as a composition for intracellular delivery of biosensors and biologically active substances.
탄소나노튜브(CNT)는 다양한 전기적, 광학적, 기계적, 생물학적 응용범위를 가지는 나노물질이다. CNT를 바이오소재로 사용하려면, 몇 가지 물리적, 화학적, 생물학적 특징이 충족되어야 한다. CNT는 본래 수용액에서 불용성이며 심하게 응집된다. 생체 시스템은 기본적으로 수용성 분자 및 구조체로 구성되어 있으므로, CNT의 생물학적 이용을 위해서는 수용액 내에서의 가용화 문제가 해결되어야 한다. 또한, CNT가 특정한 생물학적 기능을 수행할 수 있도록 하기 위해 그 표면을 생체활성 분자로 기능화하는 것이 중요하다.Carbon nanotubes (CNTs) are nanomaterials with a wide range of electrical, optical, mechanical and biological applications. To use CNTs as biomaterials, several physical, chemical and biological characteristics must be met. CNTs are essentially insoluble in aqueous solutions and heavily aggregated. Since the biological system is basically composed of water-soluble molecules and structures, the problem of solubilization in aqueous solution has to be solved for the biological use of CNTs. It is also important to functionalize the surface with bioactive molecules in order to allow CNTs to perform specific biological functions.
CNT의 가용화를 위한 두 가지 주요 접근방법은 공유결합에 의한 기능화와 비공유결합에 의한 기능화이다. 공유결합에 의한 기능화에서는 화학반응을 통해 CNT의 결함 자리에 카르복실산 또는 아민과 같은 작용기를 형성하는데, 이들 작용기는 생체활성 분자와의 컨주게이션 반응에 이용될 수 있다. 이 방법의 문제점은 CNT 고유의 구조적, 전기적 특징이 훼손될 수 있다는 점이다.Two main approaches to solubilizing CNTs are functionalization by covalent and non-covalent functionality. In functionalization by covalent bonds, chemical reactions form functional groups, such as carboxylic acids or amines, on defective sites of the CNTs, which can be used for conjugation reactions with bioactive molecules. The problem with this method is that the structural and electrical features inherent to CNTs can be compromised.
이에 비해, 비공유결합에 의한 기능화 방법에서는 CNT의 π-컨주게이션 시스템이 보존될 수 있다. 이 방법에서는 주로 양친매성 분자를 이용하는데, 양친매성 분자의 소수성 부분은 CNT의 벽을 둘러싸고 친수성 부분이 수용액과 상호작용을 한다. 저분자량 분자 또는 계면활성제, 폴리머, 탄수화물 등이 CNT의 비공유결합 기능화 및 가용화를 위해 사용되어 왔다.In contrast, π-conjugation systems of CNTs can be preserved in non-covalent functionalization methods. This method mainly uses amphiphilic molecules, where the hydrophobic portion of the amphiphilic molecule surrounds the walls of the CNT and the hydrophilic portion interacts with the aqueous solution. Low molecular weight molecules or surfactants, polymers, carbohydrates and the like have been used for non-covalent functionalization and solubilization of CNTs.
종래 기술로서, 한국공개특허(공개번호 제10-2004-0075620호)에는 고체 상태에서 비공유 결합을 통하여 탄소나노튜브/핵산 결합체를 형성한 탄소나노튜브의 기능화 내용이 기재되어 있고, 한국등록특허(등록번호 제10-0682381호)에 탄소나노튜브와 난백-단백질이 비공유결합된 탄소나노튜브 유도체에 관한 내용이 기재되어 있다.As a prior art, Korean Patent Publication (Publication No. 10-2004-0075620) describes the functionalization of carbon nanotubes in which carbon nanotubes / nucleic acid conjugates are formed through non-covalent bonds in a solid state, and a Korean registered patent ( Registration No. 10-0682381) describes a carbon nanotube derivative in which a carbon nanotube and an egg white protein are non-covalently bound.
본 발명이 해결하고자 하는 첫 번째 과제는 생체활성 및 친수성 펩티드 부분을 β-시트 부분과 컨주게이션하여 형성한 생체활성 β-시트 폴리펩티드 블록 공중합체를 이용하여 기능화한 탄소나노튜브 복합체를 제공하는 것이다.The first object of the present invention is to provide a carbon nanotube complex functionalized using a bioactive β-sheet polypeptide block copolymer formed by conjugating a bioactive and hydrophilic peptide portion with a β-sheet portion.
본 발명이 해결하고자 하는 두 번째 과제는 상기 β-시트 폴리펩티드 블록 공중합체를 이용하여 기능화한 탄소나노튜브 복합체를 활용한 바이오 센서 및 생물학적 활성 물질의 세포내 전달용 조성물을 제공하는 것이다.The second problem to be solved by the present invention is to provide a composition for intracellular delivery of a biosensor and a biologically active substance using a carbon nanotube complex functionalized using the β-sheet polypeptide block copolymer.
본 발명이 해결하고자 하는 세 번째 과제는 상기 β-시트 폴리펩티드 블록 공중합체를 이용하여 기능화한 탄소나노튜브 복합체의 제조방법을 제공하는 것이다.The third object of the present invention is to provide a method for producing a carbon nanotube complex functionalized using the β-sheet polypeptide block copolymer.
본 발명은 상기 첫 번째 과제를 달성하기 위하여,The present invention to achieve the first object,
β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체로서,As a complex of β-sheet polypeptide block copolymer and carbon nanotube,
상기 β-시트 폴리펩티드 블록 공중합체는 β-시트 폴리펩티드 블록 및 생체활성 폴리펩티드 블록으로 이루어지고,The β-sheet polypeptide block copolymer consists of a β-sheet polypeptide block and a bioactive polypeptide block,
상기 β-시트 폴리펩티드 블록은 비극성 아미노산과 극성 아미노산이 교대로 반복되는 구조, 또는 비극성 아미노산이 50-100%인 구조를 가지며,The β-sheet polypeptide block has a structure in which non-polar amino acids and polar amino acids are alternately repeated, or 50-100% of non-polar amino acids,
상기 생체활성 폴리펩티드 블록은 생체활성 폴리펩티드 블록을 이루는 아미노산의 50-100%가 극성 아미노산으로 이루어지며,The bioactive polypeptide block is composed of polar amino acids 50-100% of the amino acids constituting the bioactive polypeptide block,
상기 β-시트 폴리펩티드 블록은 상기 탄소나노튜브 표면과 비공유 결합을 하고,The β-sheet polypeptide block is non-covalently bonded to the surface of the carbon nanotubes,
상기 생체활성 폴리펩티드 블록은 친수성을 띠면서 상기 복합체 외곽으로 노출되는 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체를 제공한다.The bioactive polypeptide block is hydrophilic and provides a complex of β-sheet polypeptide block copolymer and carbon nanotube, which is exposed to the outside of the complex.
본 발명의 일 실시예에 의하면, 상기 비극성 아미노산은 페닐알라닌(phenylalanine), 알라닌(alanine), 발린(valine), 이소류신(isoleucine), 류신(leucine), 메치오닌(methionine), 티로신(tyrosine) 및 트립토판(tryptophan) 중에서 선택될 수 있다.According to one embodiment of the invention, the non-polar amino acid is phenylalanine (phenylalanine), alanine (alanine), valine (valine), isoleucine (isoleucine), leucine, methionine (methionine), tyrosine (tyrosine) and tryptophan ( tryptophan).
본 발명의 일 실시예에 의하면, 상기 극성 아미노산은 각각 독립적으로 라이신(lysine), 글라이신(glycine), 아르기닌(arginine), 프롤린(proline), 글루타민(glutamine), 세린(serine), 히스티딘(histidine), 아스파라긴산(aspartic acid), 글루타민산(glutamic acid), 트레오닌(threonine), 아스파라긴(aspargine), 시스테인(cysteine) 및 셀레노시스테인(selenocysteine) 중에서 선택될 수 있다.According to one embodiment of the invention, the polar amino acids are each independently lysine (glycine), glycine (glycine), arginine (arginine), proline (proline), glutamine (glutamine), serine, histidine (histidine) , Aspartic acid (glucomic acid), glutamic acid (glutamic acid), threonine (threonine), asparagine (aspargine), cysteine (cysteine) and selenocysteine (selenocysteine).
본 발명의 다른 일 실시예에 의하면, β-시트 폴리펩티드 블록의 비극성 아미노산은 페닐알라닌 또는 트립토판이고, 상기 극성 아미노산은 라이신, 글루탐산 또는 글라이신일 수 있다.According to another embodiment of the present invention, the non-polar amino acid of the β-sheet polypeptide block is phenylalanine or tryptophan, and the polar amino acid may be lysine, glutamic acid or glycine.
본 발명의 다른 일 실시예에 의하면, 상기 β-시트 폴리펩티드 블록은 펩티드 가닥 사이에서 β-시트 수소결합을 형성하여 자기조립을 형성하고, 상기 페닐알라닌 또는 트립토판이 상기 탄소나노튜브 표면과 π-π 스태킹 및 소수성 상호작용을 통하여 비공유 결합을 형성할 수 있다.According to another embodiment of the present invention, the β-sheet polypeptide block forms β-sheet hydrogen bonds between peptide strands to form self-assembly, and the phenylalanine or tryptophan stacks π-π with the surface of the carbon nanotubes. And non-covalent bonds through hydrophobic interactions.
본 발명의 다른 일 실시예에 의하면, 상기 β-시트 폴리펩티드 블록 공중합체는 N-말단과 C-말단이 결합된 거대고리형(macrocyclic) 펩티드일 수 있다.According to another embodiment of the present invention, the β-sheet polypeptide block copolymer may be a macrocyclic peptide having an N-terminus and a C-terminus bonded thereto.
본 발명은 상기 두 번째 과제를 달성하기 위하여,The present invention to achieve the second object,
상기 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체, 및 상기 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체에 표적 바이오 물질과 반응할 수 있는 리간드 또는 리셉터가 부착된 것을 특징으로 하는 바이오 센서를 제공한다.A ligand or a receptor capable of reacting with a target biomaterial is attached to the β-sheet polypeptide block copolymer and the carbon nanotube complex, and the β-sheet polypeptide block copolymer and the carbon nanotube complex. It provides a biosensor.
본 발명의 일 실시예에 의하면, 상기 리간드 및 리셉터는 효소기질, 리간드, 아미노산, 펩티드, 단백질, 효소, 지질, 코펙터, 탄수화물 및 이들 2종의 조합일 수 있다.According to an embodiment of the present invention, the ligand and receptor may be an enzyme substrate, a ligand, an amino acid, a peptide, a protein, an enzyme, a lipid, a cofactor, a carbohydrate, and a combination of the two.
또한, 본 발명은 상기 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체를 유효 성분으로 포함하는 생물학적 활성 물질의 세포내 전달용 조성물을 제공한다.The present invention also provides a composition for intracellular delivery of a biologically active substance comprising a complex of the β-sheet polypeptide block copolymer and a carbon nanotube as an active ingredient.
본 발명은 상기 세 번째 과제를 달성하기 위하여,The present invention to achieve the third object,
(a) 탄소나노튜브 현탁액에서 현탁 용매를 제거함으로써 전처리된 탄소나노튜브를 수득하는 단계; 및(a) obtaining a pretreated carbon nanotube by removing the suspension solvent from the carbon nanotube suspension; And
(b) 상기 전처리된 탄소나노튜브를 β-시트 폴리펩티드 블록 공중합체 수용액에 추가하여 분산액을 수득하는 단계;를 포함하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체의 제조방법을 제공한다.(b) adding the pretreated carbon nanotubes to an aqueous β-sheet polypeptide block copolymer solution to obtain a dispersion; and providing a method of preparing a complex of β-sheet polypeptide block copolymer and carbon nanotubes, including the dispersion. .
본 발명의 일 실시예에 의하면, 상기 분산액은 염을 추가로 포함할 수 있다.According to one embodiment of the invention, the dispersion may further comprise a salt.
본 발명의 다른 일 실시예에 의하면, 상기 (a) 단계의 상기 현탁 용매는 테트라 하이드로 퓨란이고, 상기 현탁 용매는 원심분리 방법에 의해서 제거될 수 있다.According to another embodiment of the present invention, the suspension solvent of step (a) is tetrahydrofuran, the suspension solvent may be removed by a centrifugation method.
본 발명의 다른 일 실시예에 의하면, 상기 염의 상기 분산액 내 농도가 1 mM 이상이고, 상기 분산액은 초음파 방법을 이용하여 제조할 수 있다.According to another embodiment of the present invention, the concentration of the salt in the dispersion is 1 mM or more, the dispersion may be prepared using an ultrasonic method.
본 발명은 생체활성을 갖는 양친매성 β-시트 폴리펩티드 블록 공중합체를 이용하여 탄소나노튜브를 기능화한 것으로서, 이에 따라 제조된 펩티드/탄소나노튜브 복합체는 수분산성이 우수하고, 생체활성을 가져서 자극 반응성 바이오 소재로서 또는 CNT 기반 전자 바이오 센서 장치의 제작에 사용될 수 있다. 또한, 생물학적 활성 물질의 세포내 전달용 조성물로서도 활용이 가능하고, β-시트 펩티드와 탄소 기반 소수성 물질 사이의 상호작용을 응용하여 단백질의 이상접힘에 의한 질환에 대한 억제제의 설계 및 개발에도 유용할 것으로 기대된다.The present invention is the functionalization of carbon nanotubes using an amphipathic β-sheet polypeptide block copolymer having a bioactivity, and the peptide / carbon nanotube complexes prepared according to the present invention are excellent in water dispersibility and have bioactivity to stimulate responsiveness. It can be used as a biomaterial or in the manufacture of CNT based electronic biosensor devices. In addition, it can be used as a composition for intracellular delivery of a biologically active substance, and may be useful for the design and development of inhibitors for diseases caused by abnormal folding of proteins by applying the interaction between β-sheet peptides and carbon-based hydrophobic substances. It is expected to be.
도 1은 본 발명에 따른 β-시트 폴리펩티드 블록 공중합체와 단일벽 탄소나노튜브(SWCNT)의 다양한 조합/자기조립의 반응 경로를 보여주는 개념도로서, 펩티드의 a 부분(GRKKRRQRRRPPQGSGG)은 생체활성 부분이고, b 부분(FKFEFKFEFKFE)은 β-시트 자기조립 부분이다.1 is a conceptual diagram showing a reaction route of various combinations / self-assembly of β-sheet polypeptide block copolymers and single-walled carbon nanotubes (SWCNTs) according to the present invention, wherein a part (GRKKRRQRRRPPQGSGG) of the peptide is a bioactive part, Part b (FKFEFKFEFKFE) is the β-sheet self-assembled part.
도 2는 순수한 물 내에서 SWCNT의 가용화를 확인한 것으로서,Figure 2 confirms the solubilization of SWCNT in pure water,
(a)는 β-시트 폴리펩티드 블록 공중합체의 농도가 SWCNT의 가용화에 미치는 영향을 확인한 이미지이고,(숫자는 펩티드의 농도를 나타낸다)(a) is an image confirming the effect of the concentration of β-sheet polypeptide block copolymer on the solubilization of SWCNT (number represents the concentration of peptide)
(b)는 동적 광산란(DLS)에 의해 측정한, 가용화된 펩티드/SWCNT 복합체의 수화반경(R H)분포를 나타낸 그래프이며,(b) is a graph showing the hydration radius ( R H ) distribution of the solubilized peptide / SWCNT complex, as measured by dynamic light scattering (DLS),
(c)는 왼쪽부터 펩티드 농도 1.5, 5 및 12.5 mM에서 β-시트 폴리펩티드 블록 공중합체의 농도가 SWCNT의 가용화에 미치는 영향을 확인한 이미지이다. (SWCNT의 양은 5 mg임)(c) is an image confirming the effect of β-sheet polypeptide block copolymer concentration on the solubilization of SWCNTs from the left at peptide concentrations 1.5, 5 and 12.5 mM. (The amount of SWCNT is 5 mg)
도 3은 이온세기가 β-시트 펩티드와 SWCNT의 조합 자기조립에 미치는 영향을 확인한 것으로서,3 shows the effect of ionic strength on the combination self-assembly of β-sheet peptide and SWCNT,
(a)는 순수한 물(왼쪽) 또는 50 mM NaCl(오른쪽) 내의, 볼텍싱한 펩티드 샘플을 나타내는 이미지이다.(a) is an image showing the vortexed peptide sample in pure water (left) or 50 mM NaCl (right).
(b)는 순수한 물(왼쪽) 또는 50 mM NaCl(오른쪽) 내의, 초음파 처리한 펩티드 샘플을 나타내는 이미지이다.(b) is an image showing the sonicated peptide sample in pure water (left) or 50 mM NaCl (right).
(c)는 CD 스펙트럼을 나타낸 그래프로서, a(굵은 실선, 5 mM 펩티드 + 60 mM NaCl), b(점선, 5 mM 펩티드 + 20 mM NaCl), c(이점쇄선, 5 mM 펩티드 + 60 mM NaCl + 5 mg SWCNT), d(가는 실선, 5 mM 펩티드 + 20 mM NaCl + 5 mg SWCNT)이다.(c) is a graph showing the CD spectrum, a (thick solid line, 5 mM peptide + 60 mM NaCl), b (dotted line, 5 mM peptide + 20 mM NaCl), c (double-stranded line, 5 mM peptide + 60 mM NaCl) + 5 mg SWCNT), d (thin solid, 5 mM peptide + 20 mM NaCl + 5 mg SWCNT).
(d)는 펩티드 단돋(b, 점선) 및 펩티드/SWCNT 복합체(a, 실선)의 형광방출 스펙트럼을 나타낸 그래프이다.(d) is a graph showing the fluorescence emission spectra of the peptide single layer (b, dotted line) and the peptide / SWCNT complex (a, solid line).
(e) 및 (f)는 각각 순수한 물 내(e) 및 20 mM NaCl 존재 하(f)의, 가용화된 펩티드/SWCNT(50 mM/5 mg) 복합체의 TEM 이미지이다.(e) and (f) are TEM images of solubilized peptide / SWCNT (50 mM / 5 mg) complexes in pure water (e) and in the presence of 20 mM NaCl (f), respectively.
도 4는 HeLa 세포에서 펩티드/SWCNT 복합체의 세포 간 전달을 확인한 이미지로서,4 is an image confirming the intercellular delivery of the peptide / SWCNT complex in HeLa cells,
(a)는 펩티드/SWCNT 복합체의 세포 내 분포를 나타내는 이미지이고,(a) is an image showing the intracellular distribution of the peptide / SWCNT complex,
(b)는 LysoTracker Red DND-99로 염색한 세포 이미지이며,(b) is a cell image stained with LysoTracker Red DND-99,
(c)는 합성 이미지이다. (배율 ×400)(c) is a composite image. (Magnification × 400)
도 5는 이온 세기가 폴리 펩티드 블록 공중합체의 β-시트 형성에 미치는 영향을 확인한 것으로서,5 confirms the effect of ionic strength on the β-sheet formation of the polypeptide block copolymer,
(a)는 여러 염 농도에서 측정한 CD 스펙트럼이다.(a) is the CD spectrum measured at various salt concentrations.
(b)는 215 nm에서 측정한 β-시트 폴리펩티드 블록 공중합체 내 β-시트 함량에 따른 분자의 타원율을 나타낸 그래프이다.(b) is a graph showing the ellipticity of molecules according to the β-sheet content in the β-sheet polypeptide block copolymer measured at 215 nm.
도 6은 이온 세기가 폴리 펩티드 블록 공중합체의 β-시트 형성에 미치는 영향을 확인한 것으로서,Figure 6 confirms the effect of ionic strength on the β-sheet formation of the polypeptide block copolymer,
(a)는 여러 염(0-120 mM) 농도에서 측정한 CD 스펙트럼이다.(a) is CD spectrum measured at various salt (0-120 mM) concentrations.
(b)는 215 nm에서 측정한 β-시트 폴리펩티드 블록 공중합체 내 β-시트 함량에 따른 분자의 타원율을 나타낸 그래프이다.(b) is a graph showing the ellipticity of molecules according to the β-sheet content in the β-sheet polypeptide block copolymer measured at 215 nm.
도 7은 본 발명에 따른 고리형 β-시트 폴리펩티드 블록 공중합체(합성예 2의 펩티드 1 내지 4)의 구조이다.Fig. 7 shows the structure of the cyclic β-sheet polypeptide block copolymer (peptides 1 to 4 of Synthesis Example 2) according to the present invention.
도 8은 본 발명에 따른 고리형(cyclic)의 폴리펩티드 블록 공중합체와 SWCNT를 결합하여 가용화 여부를 확인한 이미지로서,8 is an image confirming the solubilization by combining the cyclic polypeptide block copolymer and SWCNT according to the present invention,
좌측 이미지는 20 mM의 염 수용액 내에서, 우측 이미지는 150 mM의 염 수용액내/초음파 처리한 경우의 이미지이다.Left image in 20 mM aqueous salt solution, right image in 150 mM aqueous salt solution / ultrasonic treatment.
도 9는 가용화된 고리형 폴리펩티드/SWCNT의 TEM 이미지이다.9 is a TEM image of a solubilized cyclic polypeptide / SWCNT.
도 10은 본 발명에 따른 고리형 폴리펩티드/SWCNT 복합체의 펩티드 α-나선구조 안정화 기능을 확인하기 위하여 측정한 CD 스펙트럼이다10 is a CD spectrum measured to confirm the peptide α-helix stabilization function of the cyclic polypeptide / SWCNT complex according to the present invention
도 11은 SWCNT의 가용화를 위한 폴리펩티드 공중합체의 β-시트 형성 부분으로서 페닐알라닌 잔기 외에 트립토판 잔기의 이용 가능성을 모색하기 위해 본 발명에 따른 고리형 펩티드를 SWCNT 가용화를 확인한 이미지이다.11 is an image confirming SWCNT solubilization of a cyclic peptide according to the present invention to explore the availability of tryptophan residues in addition to phenylalanine residues as β-sheet forming portions of polypeptide copolymers for solubilization of SWCNTs.
도 12는 β-시트 폴리펩티드 부분의 β-시트 형성 잔기, 특히 트립토판 잔기의 개수와 배치를 달리하여도 SWCNT를 가용화시킬 수 있음을 확인한 이미지이다.FIG. 12 is an image showing that SWCNTs can be solubilized by varying the number and arrangement of β-sheet forming residues, particularly tryptophan residues, of the β-sheet polypeptide portion.
도 13은 본 발명에 따른 고리형 β-시트 폴리펩티드 블록 공중합체의 합성에 있어서, 고리화 반응 과정을 나타낸 개념도이다.Figure 13 is a conceptual diagram showing a cyclization process in the synthesis of cyclic β-sheet polypeptide block copolymer according to the present invention.
이하, 본 발명을 더욱 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
펩티드(단백질의 최소화된 형태)는 적절한 설계 및 자기조립을 통해 제어 가능한 나노구조를 형성하도록 함으로써 단백질 유사 인공 나노물질로 개발할 수 있다.Peptides (minimized forms of proteins) can be developed into protein-like artificial nanomaterials by forming controllable nanostructures through proper design and self-assembly.
특히, 자기조립 β-시트 펩티드는 생체활성 및 친수성 펩티드 부분을 β-시트 부분과 컨주게이션하여 생체활성 β-시트 폴리펩티드 블록 공중합체를 형성함으로써 생체활성 나노구조로의 기능화가 가능하다. 이를 통해, 제어 가능하며 생물학적으로 유용한 다양한 형태의 β-시트 펩티드 나노구조를 제조할 수 있다.In particular, self-assembled β-sheet peptides can be functionalized into bioactive nanostructures by conjugating bioactive and hydrophilic peptide moieties with β-sheet moieties to form bioactive β-sheet polypeptide block copolymers. This enables the production of various forms of controllable and biologically useful β-sheet peptide nanostructures.
β-시트 폴리펩티드 블록 공중합체는 스스로 자기조립하여 나노구조체를 형성할 수 있다. 또한, β-시트 폴리펩티드 블록 공중합체의 자기조립 현상을 제어하면 스스로 자기조립 하는 대신 탄소나노튜브와 공자기조립(co-assembly)하게 만드는 것이 가능하다.β-sheet polypeptide block copolymers can self-assemble themselves to form nanostructures. In addition, controlling the self-assembly of the β-sheet polypeptide block copolymer enables co-assembly with carbon nanotubes instead of self-assembly.
본 발명은 β-시트 폴리펩티드 블록 공중합체와 탄소나노튜브를 공자기조립하여 β-시트 폴리펩티드 블록 공중합체/탄소나노튜브 복합체를 만드는 기술을 제공하는 것이다.The present invention provides a technique of co-self-assembling β-sheet polypeptide block copolymer and carbon nanotube to make β-sheet polypeptide block copolymer / carbon nanotube complex.
본 발명은 생체활성 β-시트 폴리펩티드 블록 공중합체와 CNT가 조합되어 자기조립된 생체활성 탄소나노튜브 복합체를 제공한다.The present invention provides a self-assembled bioactive carbon nanotube complex by combining a bioactive β-sheet polypeptide block copolymer and a CNT.
생체활성 폴리펩티드와 β-시트 폴리펩티드가 공중합된 복수개의 β-시트 폴리펩티드 블록 공중합체가 비공유 결합에 의해서 탄소나노튜브 표면의 전부 또는 일부를 감싸고, 상기 β-시트 폴리펩티드 부분은 상기 탄소나노튜브 표면과 결합하고, 상기 친수성 폴리펩티드 부분은 전하를 띠면서 외곽 표면층으로 노출되며, 상기 생체활성 폴리펩티드는 라이신, 글라이신, 아르기닌, 프롤린, 글루타민 및 세린 중에서 선택되는 1종 이상의 아미노산이 복수개로 반복되어 이루어지고, 상기 β-시트 폴리펩티드는 페닐알라닌, 트립토판, 라이신, 글루탐산 및 글라이신 중에서 선택되는 1종 이상의 아미노산이 복수개로 반복되어 이루어지는 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체-탄소나노튜브 수용성 복합체를 제공한다.A plurality of β-sheet polypeptide block copolymers in which a bioactive polypeptide and a β-sheet polypeptide are copolymerized surround all or a portion of the carbon nanotube surface by non-covalent bonds, and the β-sheet polypeptide portion is bonded to the carbon nanotube surface. The hydrophilic polypeptide portion is charged and exposed to the outer surface layer, and the bioactive polypeptide is composed of a plurality of repeating one or more amino acids selected from lysine, glycine, arginine, proline, glutamine and serine, and the β The -sheet polypeptide provides a β-sheet polypeptide block copolymer-carbon nanotube water-soluble complex, characterized in that a plurality of one or more amino acids selected from phenylalanine, tryptophan, lysine, glutamic acid and glycine are repeated in plurality.
상기 β-시트 폴리펩티드 블록 공중합체는 선형(linear) 펩티드이거나, N-말단과 C-말단이 결합된 거대고리형(macrocyclic) 펩티드인 것을 특징으로 한다.The β-sheet polypeptide block copolymer may be a linear peptide or a macrocyclic peptide having an N-terminus and a C-terminus bonded thereto.
본 발명에 따른 자기조립 시스템에서는 두 가지의 경쟁적인 힘, 즉 β-시트 부분 사이의 인력과 β-시트 부분 및 CNT 사이의 인력이 균형을 이루어야 한다. 전자가 우세할 경우, 펩티드는 β-시트 나노구조의 형성에 참여하지만 CNT는 수용액 내에 불용 상태로 남게 된다. 한편, 후자가 우세할 경우, 생체활성 β-시트 폴리펩티드 블록 공중합체에 의해 비공유적으로 기능화된 수용성/생체활성 CNT가 얻어질 수 있다. 또한, 상기 경쟁적인 힘들 사이의 균형을 보다 상세하게 규명할 경우에 단백질의 이상접힘에 의한 질환에 있어 β-아밀로이드 및 관련 단백질의 제어 및 응집방지가 가능할 수도 있다.In the self-assembly system according to the present invention, two competing forces, i.e., the attraction between the β-sheet portion and the attraction between the β-sheet portion and the CNT, must be balanced. If the former dominates, the peptide participates in the formation of β-sheet nanostructures but the CNTs remain insoluble in the aqueous solution. On the other hand, when the latter prevails, water-soluble / bioactive CNTs that are non-covalently functionalized by bioactive β-sheet polypeptide block copolymers can be obtained. In addition, when the balance between the competitive forces is elucidated in detail, it may be possible to control and prevent aggregation of β-amyloid and related proteins in diseases caused by abnormal folding of proteins.
상기 β-시트 폴리펩티드는 펩티드 가닥 사이에서 β-시트 수소결합을 형성하여 자기조립되는 것을 특징으로 하고, 상기 β-시트 폴리펩티드의 페닐알라닌 또는 트립토판 잔기가 π-π 스태킹 상호작용을 통하여 상기 탄소나노튜브 표면과 결합하는 것을 특징으로 한다.The β-sheet polypeptides are self-assembled by forming β-sheet hydrogen bonds between peptide strands, and the phenylalanine or tryptophan residues of the β-sheet polypeptides interact with each other through a π-π stacking interaction. It is characterized by combining with.
이와 같이, β-시트 폴리펩티드 블록 공중합체는 극성과 응집경향에 있어 매우 다른 성격을 갖는 두 부분으로 구성된다.As such, the β-sheet polypeptide block copolymer consists of two parts having very different characteristics in polarity and aggregation tendency.
생체활성 부분(예를 들면, 하기 도 1의 a 부분)은 세포 투과성 펩티드인 Tat를 기본으로 한다. 이 부분은 다수의 라이신 및 아르기닌을 포함하므로, 수용액 내에서 높은 전하를 띠며 극성을 갖게 된다. 자기조립 부분은 비극성 아미노산(페닐알라닌 또는 트립토판)과 극성 아미노산(라이신 및 글루탐산)이 반복되는 구조를 가지며, 펩티드 가닥 사이의 β-시트 수소결합 형성을 촉진한다. 이러한 구조를 갖는 폴리펩티드 블록 공중합체가 친수성 부분의 큰 부피와 다수의 양전하 때문에 충분히 높은 이온세기 하에서만 β-시트 나노리본 구조를 형성할 수 있다.(하기 도 1의 경로 1). 염이 존재하지 않거나 이온세기가 낮은 경우, 폴리펩티드 블록 공중합체는 주로 랜덤코일의 형태로 존재한다.The bioactive moiety (eg, part a of FIG. 1 below) is based on Tat, a cell permeable peptide. This part contains a large number of lysine and arginine, so it is highly charged and polarized in aqueous solution. The self-assembled moiety has a repeating structure of nonpolar amino acids (phenylalanine or tryptophan) and polar amino acids (lysine and glutamic acid) and promotes β-sheet hydrogen bond formation between peptide strands. Polypeptide block copolymers having this structure can form β-sheet nanoribbon structures only under sufficiently high ionic strength because of the large volume of the hydrophilic moiety and the large number of positive charges (Path 1 in FIG. 1 below). If no salt is present or the ionic strength is low, the polypeptide block copolymer is mainly in the form of a random coil.
본 발명은 상기 β-시트 폴리펩티드 블록 공중합체-탄소나노튜브 수용성 복합체에 표적 바이오 물질과 반응하는 리간드 또는 리셉터를 부착시킨 것을 특징으로 하는 바이오 센서를 제공한다.The present invention provides a biosensor comprising attaching a ligand or a receptor reacting with a target biomaterial to the β-sheet polypeptide block copolymer-carbon nanotube-soluble complex.
본 발명에 있어서, 표적 바이오물질 혹은 유기화합물은 리간드 또는 리셉터와 반응하여 검출되는 표적 역할을 할 수 있는 물질로서, 바람직하게는 단백질, 핵산, 항체, 효소, 탄수화물, 지질 또는 기타 바이오 분자이며, 더욱 바람직하게는 질병에 관련된 단백질, 핵산, 그리고 탄수화물이다.In the present invention, the target biomaterial or organic compound is a substance capable of serving as a target to be detected by reacting with a ligand or receptor, preferably a protein, nucleic acid, antibody, enzyme, carbohydrate, lipid or other biomolecule, and more. Preferably proteins, nucleic acids, and carbohydrates associated with the disease.
본 발명에 있어서, 리간드 또는 리셉터는 효소기질, 리간드, 아미노산, 펩타이드, 단백질, 핵산, 지질, 코펙터 또는 탄수화물인 것을 특징으로 할 수 있다.In the present invention, the ligand or receptor may be characterized in that the enzyme substrate, ligand, amino acids, peptides, proteins, nucleic acids, lipids, cofactors or carbohydrates.
또한, 본 발명은 β-시트 폴리펩티드 블록 공중합체-탄소나노튜브 수용성 복합체를 유효 성분으로 포함하는 생물학적 활성 물질의 세포내 전달용 조성물을 제공한다.The present invention also provides a composition for intracellular delivery of a biologically active substance comprising a β-sheet polypeptide block copolymer-carbon nanotube water-soluble complex as an active ingredient.
본 발명의 조성물은 종래에 생물학적 활성물질이 쉽게 통과할 수 없었던 세포막을 통과하여 세포 내에 직접 작용할 수 있게 한다. 따라서 본 발명의 조성물은 약물전달체계(drug delivery system)의 발전에 획기적인 계기가 될 수 있다.The composition of the present invention allows the biologically active substance to act directly in the cell through the cell membrane, which has not been able to easily pass through. Therefore, the composition of the present invention can be a breakthrough in the development of drug delivery system (drug delivery system).
본 발명의 조성물은, 조성물 총 중량에 대하여 상기 유효성분을 0.0001 내지 50 중량%로 포함한다.The composition of the present invention comprises 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition.
본 발명의 조성물은 상기 유효성분에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다.The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.
본 발명의 조성물은, 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올, 리포좀 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있으며, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다. 더 나아가 당해 기술분야의 적정한 방법으로 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as needed. And other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art.
상기 융합체를 유효성분으로 포함하는 조성물은 정맥내(intravein), 복막내(intraperitoneal), 근육내(intramuscular), 피하내(subcutaneous), 피내(intradermal), 비내(nasal), 점막내(mucosal), 흡입(inhalation) 및 경구(oral) 등의 경로로 주입함으로써 생체 내 전달될 수 있다. 투여량은 대상의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 일일 투여량은 화합물의 경우 약 0.1 내지 100 ㎎/㎏이고, 바람직하게는 0.5 내지 10 ㎎/㎏이며, 하루 일회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다.The composition comprising the fusion as an active ingredient is intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, nasal, mucosal, mucosal, It can be delivered in vivo by infusion, such as by inhalation and oral. Dosage varies depending on the subject's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of disease. The daily dosage is about 0.1 to 100 mg / kg for the compound, preferably 0.5 to 10 mg / kg, and more preferably administered once to several times a day.
본 발명은 (a) 탄소나노튜브를 테트라하이드로퓨란에 현탁시킨 후, 원심분리하여 테트라하이드로퓨란을 증발시켜 전처리된 탄소나노튜브를 수득하는 단계 및 (b) 상기 전처리한 탄소나노튜브, 5-200 μM β-시트 폴리펩티드 블록 공중합체 수용액 및 10-150 mM 염화나트륨 수용액을 혼합하여 초음파처리하는 단계를 포함하는 β-시트 폴리펩티드 블록 공중합체-탄소나노튜브 수용성 복합체의 제조방법을 제공한다.The present invention comprises the steps of (a) suspending carbon nanotubes in tetrahydrofuran and centrifuging to evaporate tetrahydrofuran to obtain pretreated carbon nanotubes, and (b) the pretreated carbon nanotubes, 5-200. It provides a method for producing a β-sheet polypeptide block copolymer-carbon nanotube water-soluble complex comprising the step of mixing and sonicating the μM β-sheet polypeptide block copolymer aqueous solution and 10-150 mM sodium chloride aqueous solution.
상기 β-시트 폴리펩티드 블록 공중합체는 생체활성 폴리펩티드와 β-시트 폴리펩티드가 블록 공중합된 것이며, 상기 생체활성 폴리펩티드는 라이신, 글라이신, 아르기닌, 프롤린, 글루타민 및 세린 중에서 선택되는 1종 이상의 아미노산이 복수개로 반복되어 이루어지고, 상기 β-시트 폴리펩티드는 페닐알라닌, 트립토판, 라이신, 글루탐산 및 글라이신 중에서 선택되는 1종 이상의 아미노산이 복수개로 반복되어 이루어지는 것을 특징으로 한다.The β-sheet polypeptide block copolymer is a block copolymer of a bioactive polypeptide and a β-sheet polypeptide, and the bioactive polypeptide is one or more amino acids selected from lysine, glycine, arginine, proline, glutamine and serine in a plurality of repetitions. The β-sheet polypeptide is characterized in that a plurality of repeating one or more amino acids selected from phenylalanine, tryptophan, lysine, glutamic acid and glycine.
이하, 바람직한 실시예를 들어 본 발명을 더욱 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이에 의하여 제한되지 않는다는 것은 당업계의 통상의 지식을 가진 자에게 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to preferred examples. However, these examples are intended to illustrate the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited thereby.
이하의 실시예에서, Fmoc-아미노산은 Novabiochem(독일)에서 구입하여 사용하였고, Fmoc-21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid(Fmoc-NH-PEG5-COOH)와 N-(Fmoc-8-amino-3,6-dioxaoctyl)succinamic acid (Fmoc-PEG2-Suc-OH 또는 Fmoc-Ebes-OH)는 각각 merck(독일)와 Anaspec(미국)에서 구입하였다. 아크방전법에 의해 제조된 단일벽 탄소 나노튜브(ASP-100F, "정제" 등급)는 한화나노테크(한국)에서 구입하였다. 조직배양시약은 Invitrogen(미국)에서 구입하였다.In the examples below, Fmoc-amino acids were purchased from Novabiochem (Germany) and used with Fmoc-21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid (Fmoc-NH-PEG5-COOH). N- (Fmoc-8-amino-3,6-dioxaoctyl) succinamic acid (Fmoc-PEG2-Suc-OH or Fmoc-Ebes-OH) was purchased from merck (Germany) and Anaspec (USA), respectively. Single-walled carbon nanotubes (ASP-100F, "refined" grades) prepared by the arc discharge method were purchased from Hanwha Nanotech (Korea). Tissue culture reagents were purchased from Invitrogen (USA).
합성예 1. 선형(linear) β-시트 폴리펩티드 블록 공중합체Synthesis Example 1 Linear β-Sheet Polypeptide Block Copolymer
(1) 합성 Rink Amide MBHA 레진 LL(Novabiochem) 상에서 표준 Fmoc 프로토콜에 따라 Tribute™ 펩티드 합성기(Protein Technologies, Inc)를 사용하여 펩티드를 합성하였고, 합성 과정에서 표준 아미노산 보호기를 사용하였다. 합성한 β-시트 폴리펩티드 블록 공중합체의 구성은 다음과 같다.(1) Synthesis Peptides were synthesized using Tribute ™ Peptide Synthesizer (Protein Technologies, Inc) according to standard Fmoc protocol on Rink Amide MBHA Resin LL (Novabiochem), and standard amino acid protecting groups were used during the synthesis. The structure of the synthesized β-sheet polypeptide block copolymer is as follows.
Gly-Arg-Lys-Lys-Arg-Arg-Glu-Arg-Arg-Arg-Pro-Pro-Glu-Ser-Gly-Gly-Phe-Lys-Phe-Glu-Phe-Lys-Phe-Glu-Phe-Lys-Phe-GluGly-Arg-Lys-Lys-Arg-Arg-Glu-Arg-Arg-Arg-Pro-Pro-Glu-Ser-Gly-Gly-Phe-Lys-Phe-Glu-Phe-Lys-Phe-Glu-Phe- Lys-Phe-Glu
또는 GRKKRRQRRRPPQGSGGFKFEFKFEFKFEOr GRKKRRQRRRPPQGSGGFKFEFKFEFKFE
(2) N-말단이 형광 표지된 펩티드의 합성을 위해, 1 mL의 N-메틸-2-피롤리돈(NMP)에 용해된 5-카르복시플루오레신(50 mmol), HBTU(45 mmol) 및 DIPEA(100 mmol)를 레진에 결합된 펩티드에 가하고 밤새 반응시켰다. 이어, 레진을 NMP와 아세토니트릴로 세척하고 진공 하에서 건조하였다. 건조된 레진을 분리용액(TFA:TIS:물 = 95:2.5:2.5)으로 3 시간 동안 처리한 다음, t-부틸 메틸 에테르를 이용하여 분말화하였다. 얻어진 펩티드를 역상 HPLC(물-아세토니트릴, 0.1% TFA)로 정제하였다.(2) 5-carboxyfluorescein (50 mmol), HBTU (45 mmol) dissolved in 1 mL of N-methyl-2-pyrrolidone (NMP) for the synthesis of N-terminally fluorescently labeled peptides And DIPEA (100 mmol) was added to the peptide bound to the resin and allowed to react overnight. The resin was then washed with NMP and acetonitrile and dried under vacuum. The dried resin was treated with separation solution (TFA: TIS: water = 95: 2.5: 2.5) for 3 hours and then powdered with t-butyl methyl ether. The resulting peptide was purified by reverse phase HPLC (water-acetonitrile, 0.1% TFA).
MALDI-TOF 질량분석을 통해 분자량을 측정하였다. HPLC 분석 결과 펩티드의 순도는 95% 이상이었다.The molecular weight was determined by MALDI-TOF mass spectrometry. HPLC analysis showed that the purity of the peptide was at least 95%.
물:아세토니트릴 (1:1) 내에서, 페닐알라닌의 몰흡광계수(195 m-1cm-1)를 이용하여 257.5 nm에서 농도를 분광학적으로 측정하였다.In water: acetonitrile (1: 1), the concentration was spectroscopically measured at 257.5 nm using the molar extinction coefficient of phenylalanine (195 m −1 cm −1 ).
실험예 : 선형(linear) β-시트 폴리펩티드 블록Experimental Example: Linear β-Sheet Polypeptide Block
(1) 원편광 이색성(CD)(1) circularly polar dichroism (CD)
펠티에 온도조절장치(Applied Photophysics., Ltd)가 구비된 Chirascan™ 원편광 이색성 분광계를 사용하여 CD 스펙트럼을 측정하였다. 스펙트럼은 2 mm 경로길이의 큐벳을 사용하여 250 nm에서부터 190 nm까지 측정하였다. 5회 반복 측정하여 평균값을 기록하였다. 각 아미노산 잔기에 대하여 몰 타원율을 계산하였다. 측정에 앞서, SWCNT가 포함된 또는 포함되지 않은 5 μM 펩티드 용액을 20 mM 또는 60 mM NaCl로 실온에서 12 시간 동안 처리하였다.CD spectra were measured using a Chirascan ™ circular dichroism spectrometer equipped with a Peltier thermostat (Applied Photophysics., Ltd). Spectra were measured from 250 nm to 190 nm using cuvettes of 2 mm path length. The average value was recorded by measuring 5 times repeatedly. The mole ellipticity was calculated for each amino acid residue. Prior to the measurement, 5 μM peptide solution with or without SWCNTs was treated with 20 mM or 60 mM NaCl for 12 hours at room temperature.
(2) 투과전자현미경 분석(TEM)(2) Transmission Electron Microscopy (TEM)
2 mL의 샘플을 탄소 코팅한 구리격자 상에서 완전히 건조시켰다. 이어, 2 mL의 물을 1 분간 가하여 결합되지 않은 펩티드를 용해시키고 여과지를 이용하여 제거하였다. JEOL-JEM 2010 기기를 사용하여 150 kV에서 샘플을 관찰하였다. 얻어진 데이터를 Digital Micrograph ™ 소프트웨어를 이용하여 분석하였다.2 mL of sample was completely dried over a carbon coated copper grid. 2 mL of water was then added for 1 min to dissolve the unbound peptide and removed using filter paper. Samples were observed at 150 kV using a JEOL-JEM 2010 instrument. The data obtained were analyzed using Digital Micrograph ™ software.
(3) 동적 광산란(DLS)(3) Dynamic Light Scattering (DLS)
DLS 실험은 632.8 nm의 He-Ne 레이저가 구비된 ALV/CGS-3 Compact Goniometer System을 사용하여 실온에서 수행하였다. 검출에는 광섬유가 연결된 ALV/SO-SIPD/DUAL 검출장치, EMI PM-28B 전원장치 및 ALV/PM-PD 프리앰프/판별장치를 사용하였다. 신호 분석장치로는 지수적으로 배치된 288 개의 채널을 구비한 ALV-5000/E/WIN 멀티플타우 디지털 코릴레이터를 사용하였다. 산란각은 90°였다. 크기분포는 강제조정법에 의해 결정하였다.DLS experiments were performed at room temperature using an ALV / CGS-3 Compact Goniometer System equipped with a 632.8 nm He-Ne laser. For detection, an ALV / SO-SIPD / DUAL detector with an optical fiber, an EMI PM-28B power supply, and an ALV / PM-PD preamplifier / identifier were used. The signal analyzer was an ALV-5000 / E / WIN multiple tower digital correlator with 288 exponentially arranged channels. The scattering angle was 90 degrees. The size distribution was determined by the forced adjustment method.
(4) 형광 스펙트럼분석(4) fluorescence spectrum analysis
Hitachi F-4500 형광분광계와 1 cm 경로길이의 석영 큐벳을 사용하여 정상상태 형광스펙트럼을 기록하였다. 50 mM NaCl 용액 내의 펩티드 농도는 5 mM으로 하였다. 페닐알라닌 잔기로부터 방출되는 형광을 측정하기 위하여, 샘플을 257 nm에서 여기시켰다. 여기용 및 방출용 슬릿은 공칭 밴드패스가 10 nm인 것을 사용하였다.Steady state fluorescence spectra were recorded using a Hitachi F-4500 fluorescence spectrometer and a 1 cm path length quartz cuvette. Peptide concentration in 50 mM NaCl solution was 5 mM. To measure the fluorescence emitted from the phenylalanine residues, the samples were excited at 257 nm. As the excitation and emission slits, a nominal bandpass of 10 nm was used.
(5) 조직배양 및 세포 내 전달 실험(5) Tissue culture and intracellular delivery experiment
β-시트 폴리펩티드 블록 공중합체/SWCNT 복합체의 세포 내 전달을 현미경으로 관찰하기 위하여, HeLa 세포(4×105)를 8-웰 Lab-tek Ⅱ 챔버 커버글래스 시스템(Nunc)에 분주하고 10% FBS을 포함하는 DMEM을 사용하여 37 ℃에서 밤새 배양하였다. 세포를 DPBS로 세척한 다음, 펩티드/SWCNT 복합체로 20분 동안 처리하였다. 이어, 샘플 용액을 제거하고, DMEM 내에서 90분 동안 세포를 더 배양하였다. 이미지 촬영에 앞서, 50 nM 농도의 LysoTracker Red DND-99(Invitrogen)를 5분간 가하였다. 아르곤(488 nm) 및 헬륨-네온(543 nm) 레이저가 구비된 Nikon Eclipse TE2000-U 도립현미경을 사용하여 공초점 이미지를 촬영하였다.To microscopically observe intracellular delivery of the β-sheet polypeptide block copolymer / SWCNT complex, HeLa cells (4 × 10 5 ) were dispensed into an 8-well Lab-tek II chamber cover glass system (Nunc) and 10% FBS It was incubated overnight at 37 ℃ using DMEM containing. Cells were washed with DPBS and then treated with peptide / SWCNT complex for 20 minutes. The sample solution was then removed and the cells were further incubated for 90 minutes in DMEM. Prior to imaging, LysoTracker Red DND-99 (Invitrogen) at 50 nM concentration was added for 5 minutes. Confocal images were taken using a Nikon Eclipse TE2000-U inverted microscope equipped with argon (488 nm) and helium-neon (543 nm) lasers.
실험예 1.Experimental Example 1.
먼저, β-시트를 형성할 수 있는 이 펩티드가 SWCNT와 결합하여 순수한 물 내에서 SWCNT를 가용화할 수 있는지 확인하였다.First, it was confirmed that this peptide capable of forming β-sheets could bind to SWCNTs solubilizing SWCNTs in pure water.
아크방전법을 통해 제조한 SWCNT를 테트라하이드로퓨란(THF)에 현탁하고 동량(5 mg)의 SWCNT를 마이크로 원심분리튜브에 가하였다. THF가 증발한 후, 폴리펩티드 블록 공중합체 용액(0.3 mL)을 가하고, 그 혼합물을 실온에서 15 분 동안 초음파 처리하였다.SWCNT prepared by the arc discharge method was suspended in tetrahydrofuran (THF) and the same amount (5 mg) of SWCNT was added to the microcentrifuge tube. After THF evaporated, the polypeptide block copolymer solution (0.3 mL) was added and the mixture was sonicated for 15 minutes at room temperature.
하기 도 2(a)와 2(b)에서 보는 바와 같이, 펩티드의 농도가 증가함에 따라 SWCNT가 가용화되었다. 펩티드 농도가 약 12.5 mM에 이르자 대부분의 SWCNT가 현탁되었다(하기 도2(c)). 이러한 결과로부터, 폴리펩티드 블록 공중합체의 페닐알라닌 잔기가 소수성 및 π-π 스태킹 상호작용을 통해 SWCNT와 결합하고, 라이신과 아르기닌 함량이 높은 친수성 부분은 수용액과 상호작용하여 펩티드/SWCNT 복합체의 용해를 돕는다는 것을 알 수 있다(하기 도 1).As shown in FIGS. 2 (a) and 2 (b) below, SWCNTs were solubilized with increasing peptide concentration. When the peptide concentration reached about 12.5 mM, most of the SWCNTs were suspended (Fig. 2 (c) below). From these results, the phenylalanine residue of the polypeptide block copolymer binds to SWCNT through hydrophobic and π-π stacking interactions, and the hydrophilic moiety with high content of lysine and arginine interacts with the aqueous solution to help dissolve the peptide / SWCNT complex. It can be seen that (Fig. 1 below).
또한, 제타포텐셜(ζ) 측정 결과, 펩티드/SWCNT 복합체는 큰 양의 값(+58±3 mV)을 가지는 것으로 나타났는데, 이로부터 펩티드/SWCNT 복합체 외곽층의, 양전하를 띠는 친수성 부분이 노출되어 양전하를 갖는 표면이 형성됨을 알 수 있다.In addition, zeta potential (ζ) measurements showed that the peptide / SWCNT complex had a large value (+ 58 ± 3 mV), from which the positively charged hydrophilic portion of the outer layer of the peptide / SWCNT complex was exposed. It can be seen that a surface having a positive charge is formed.
실험예 2.Experimental Example 2.
이어, 이온 세기가 펩티드/SWCNT 복합체의 형성에 미치는 영향을 확인하였다.Next, the effect of ionic strength on the formation of the peptide / SWCNT complex was confirmed.
NaCl의 농도를 높이자 폴리펩티드 블록 공중합체 용액의 β-시트 함량이 증가하였으며, 염농도 약 50-60 mM에서 증가가 멈추었다(하기 도 5 참고). 이러한 결과를 바탕으로, 이온 세기가 SWCNT의 가용화에 미치는 영향을 조사하였다. 펩티드의 농도는 순수한 물 내에서 5 mg의 SWCNT를 가용화기에는 불충분한 양인 5 mM으로 하여 실험하였다(하기 도 2(c)의 가운데 이미지).Increasing the NaCl concentration increased the β-sheet content of the polypeptide block copolymer solution and stopped increasing at a salt concentration of about 50-60 mM (see Figure 5 below). Based on these results, the effect of ionic strength on the solubilization of SWCNTs was investigated. Peptide concentrations were tested with 5 mg of SWCNT in pure water at 5 mM, an insufficient amount in the solubilizer (center image in Figure 2 (c) below).
하기 도 3(a)(왼쪽)에서 보듯이, 펩티드(5 mM)와 SWCNT의 혼합물은 순수한 물에서 큰 과립 형태의 SWCNT를 보였는데, 이로써 가용화가 불충분하다는 것을 알 수 있다. 염을 가하고(50 mM) 용액을 볼텍싱하자, 과립의 크기가 작아졌다(하기 도 3(a)의 오른쪽). 두 혼합물을 초음파 처리하자 큰 차이를 보였다. 순수한 물 내의 혼합물의 경우 공기-물 계면에서 크기가 큰 불용성 SWCNT 응집체가 형성된 반면(하기 도 3(b)의 왼쪽), 염 존재 하의 SWCNT는 거의 완전히 가용화되었다(하기 도 3(b)의 오른쪽).As shown in FIG. 3 (a) (left), the mixture of peptide (5 mM) and SWCNT showed large granular SWCNTs in pure water, indicating insufficient solubilization. Salt was added (50 mM) and the solution vortexed, and the granules became smaller (right side of Figure 3 (a) below). Ultrasonicating the two mixtures showed a big difference. In the case of mixtures in pure water, large insoluble SWCNT aggregates were formed at the air-water interface (left side of FIG. 3 (b)), while SWCNT in the presence of salt was almost completely solubilized (right side of FIG. 3 (b)). .
따라서, 이러한 결과로부터 이온세기가 높을 경우 SWCNT의 가용화가 촉진됨을 알 수 있다. 폴리펩티드 블록 공중합체가 염농도 50 mM에서 안정한 β-시트 나노리본 구조를 형성하는 것을 감안할 때(하기 도 5), β-시트 부분과 SWCNT 사이의 인력이 β-시트 부분들 사이의 인력보다 강하며, 펩티드/SWCNT 조합 자기조립 과정이 β-시트의 형성보다 빠르게 진행된다는 것을 알 수 있다. 펩티드 및 펩티드/SWCNT 복합체에 대한 CD 스펙트럼 측정결과도 이를 뒷받침한다. 215 nm에서 타원율의 음의 최대값이 크게 감소하였는데, 이로부터 SWCNT의 존재가 β-시트의 형성을 방해함을 알 수 있다(하기 도 3(c)). 복합체가 형성되면서 페닐알라닌의 형광피크가 사라진 점 역시, β-시트 부분과 SWCNT 사이의 강한 인력과 펩티드가 SWCNT에 직접 결합한다는 것을 확인시켜 준다(하기 도3(d)).Therefore, it can be seen from this result that the solubilization of SWCNT is promoted when the ionic strength is high. Given that the polypeptide block copolymers form a stable β-sheet nanoribbon structure at a salt concentration of 50 mM (FIG. 5), the attractive force between the β-sheet portion and the SWCNT is stronger than the attractive force between the β-sheet portions, It can be seen that the peptide / SWCNT combination self-assembly process proceeds faster than the β-sheet formation. CD spectral measurements of peptides and peptide / SWCNT complexes also support this. At 215 nm, the negative maximum of the ellipticity was greatly reduced, indicating that the presence of SWCNTs interfered with the formation of β-sheets (FIG. 3 (c)). The fluorescence peak of the phenylalanine disappeared as the complex was formed, confirming that the strong attraction between the β-sheet portion and the SWCNT and the peptide directly bind to the SWCNT (FIG. 3 (d)).
실험예 3.Experimental Example 3.
TEM 이미지로부터 이온세기가 펩티드/SWCNT 복합체의 형성에 미치는 영향을 더욱 분명하게 확인하였다.From the TEM image, the effect of ionic strength on the formation of the peptide / SWCNT complex was more clearly confirmed.
순수한 물 내에서 SWCNT가 안정한 상태로 잘 분산된 것처럼 보였지만, 형성된 복합체는 조건에 따라서 SWCNT 응집을 보일 수도 있음을 확인하였다(하기 도 3(e), 하기 도 1의 경로 2). 이와는 대조적으로, 염 존재 하에서 복합체를 형성한 경우, 대부분의 SWCNT가 응집되지 않은 상태였다(하기 도 3(f), 하기 도 1의 경로 3).Although SWCNTs appeared to be well dispersed in a stable state in pure water, it was confirmed that the formed complex may show SWCNT aggregation depending on the conditions (FIG. 3 (e), route 2 in FIG. 1). In contrast, when the complex was formed in the presence of salt, most of the SWCNTs were not aggregated (FIG. 3 (f) below, route 3 of FIG. 1 below).
따라서, 염의 존재가 페닐알라닌과 SWCNT 사이의 소수성 상호작용을 더욱 강화시키고 펩티드들 사이의 비특이적, 전기적 상호작용을 차단하여, SWCNT의 효과적인 가용화 및 응집 억제에 기여함을 알 수 있다.Thus, it can be seen that the presence of salt further enhances the hydrophobic interaction between phenylalanine and SWCNT and blocks the nonspecific, electrical interaction between peptides, contributing to the effective solubilization and aggregation inhibition of SWCNTs.
실험예 4.Experimental Example 4.
이미 조립된 β-시트 나노리본이 SWCNT 존재 하에 분해되어 CNT를 가용화할 수 있는지를 조사하였다(하기 도 1의 경로 4, 5).It was investigated whether the already assembled β-sheet nanoribbons could be degraded in the presence of SWCNTs to solubilize CNTs ( paths 4 and 5 in FIG. 1 below).
그 결과, β-시트 나노리본이 이미 형성된 후에는, 충분한 초음파 처리를 한 후에도 SWCNT를 펩티드로 가용화 및 기능화할 수 없음을 확인하였다.As a result, it was confirmed that after β-sheet nanoribbons were already formed, SWCNTs could not be solubilized and functionalized with peptide even after sufficient sonication.
상기의 실험예를 종합하면, β-시트 펩티드와 CNT의 조합 자기조립 과정에서 두 가지 중요한 사실을 알 수 있다.Taken together, the experimental examples show two important facts in the process of self-assembly of β-sheet peptide and CNT.
첫째로, β-시트 펩티드들 사이의 결합이 약하거나 펩티드가 자기조립의 초기단계에 있을 경우, 하기 도 1의 경로 2와 3을 조합함으로써 β-시트 폴리펩티드 블록 공중합체로 기능화된 생체활성 CNT를 제조할 수 있다.First, if the binding between β-sheet peptides is weak or the peptide is in the early stages of self-assembly, bioactive CNTs functionalized with β-sheet polypeptide block copolymers by combining pathways 2 and 3 of FIG. It can manufacture.
둘째로, 단백질의 이상접힘에 의한 질환에서 나타나는 아밀로이드 섬유의 형성을 억제하는 방안을 모색할 수 있을 것이다. 즉, 탄소의 동소체(예컨대, CNT와 풀러렌)가 아밀로이드의 형성을 억제할 수 있는데, 이미 조립된 β-시트 나노리본을 분해하는 것(경로 4, 5)이 어렵다는 점을 감안할 때, 탄소 기반 아밀로이드 억제제 개발의 주된 타겟은 아밀로이드 섬유 형성의 초기단계에서 생성되는 원섬유종과 같은 중간조립체가 될 것이다.Second, it may be possible to find ways to suppress the formation of amyloid fibers in diseases caused by abnormal folding of proteins. That is, allotropes of carbon (eg, CNTs and fullerenes) can inhibit the formation of amyloid, considering that it is difficult to decompose the already assembled β-sheet nanoribbons (paths 4 and 5). The main target of inhibitor development will be intermediate assemblies such as fibrillar species produced in the early stages of amyloid fiber formation.
실험예 5.Experimental Example 5.
경로 3에 의해 제조된 펩티드/SWCNT 복합체의 세포전달물질로의 이용 가능성을 모색하기 위하여, 포유동물 세포 내에서 복합체의 상호작용을 조사하였다.To explore the availability of peptide / SWCNT complexes prepared by pathway 3 as cell transporters, the interaction of the complexes in mammalian cells was investigated.
복합체는 세포 투과성 펩티드인 Tat로 수식되어 있으므로 세포 내로 쉽게 진입한다. Tat는 세포의 세포막과 핵막을 통과한다. 복합체를 가시화하기 위하여, 플로오레신으로 표지한 펩티드와 표지하지 않은 펩티드를 1 : 50의 몰비로 혼합하고, 그 펩티드 혼합물을 이용하여 SWCNT를 기능화하였다.The complex is modified with Tat, a cell permeable peptide, so it enters the cell easily. Tat passes through cell membranes and nuclear membranes. To visualize the complex, peptides labeled with fluorescein and unlabeled peptides were mixed in a molar ratio of 1:50 and the peptide mixture was used to functionalize SWCNTs.
하기 도 4(a)에서 보듯이, 복합체는 세포질 전체에 걸쳐 분포되어 있었다. 따라서, 복합체가 세포 내로 효과적으로 전달됨을 알 수 있다.As shown in Figure 4 (a), the complex was distributed throughout the cytoplasm. Thus, it can be seen that the complex is effectively delivered into the cell.
복합체의 녹색 형광과 LysoTracker(살아있는 세포의 산성 소기관 표지에 이용되는 프로브)의 적색 형광으로부터, 엔도시토시스 작용에 의해 복합체가 세포 내로 진입하는 것을 알 수 있다(하기 도 4(c)). Tat 펩티드는 단독으로도 세포질 내뿐 아니라 핵 이동 작용에 의해 핵과 인 안으로 진입할 수 있다. 그러나, Tat 펩티드의 핵 이동 작용에도 불구하고 복합체가 주로 세포질에 분포한다는 것은 복합체의 크기가 커서 핵공복합체(NPC)를 통과하지 못하는 것일 수 있음을 시사한다. 이러한 결과로부터 펩티드가 세포 내에서 SWCNT에 결합된 상태로 존재한다는 것을 알 수 있는데, 이것은 복합체가 안정하다는 것을 의미한다.From the green fluorescence of the complex and the red fluorescence of LysoTracker (probe used for labeling acid organelles of living cells), it can be seen that the complex enters the cell by endocytosis (Fig. 4 (c)). Tat peptides alone can enter the nucleus and phosphorus not only in the cytoplasm but also by nuclear transfer action. However, despite the nuclear transfer action of the Tat peptide, the distribution of the complex mainly in the cytoplasm suggests that the complex may be large and unable to pass through the nuclear complex (NPC). From these results it can be seen that the peptide is present in the cell bound to the SWCNT, which means that the complex is stable.
합성예 2. 고리형(cyclic) β-시트 폴리펩티드 블록 공중합체Synthesis Example 2 Cyclic β-Sheet Polypeptide Block Copolymer
(1) 합성 Rink Amide MBHA 레진 LL(Novabiochem) 상에서 표준 Fmoc 프로토콜에 따라 Tribute™ 펩티드 합성기(Protein Technologies, Inc)를 사용하여 펩티드를 합성하였고, 합성과정에서 methoxytrytil(Mmt) 보호기가 사용된 Cysteine을 제외한 나머지 아미노산에 표준 아미노산 보호기를 사용하였다. 합성한 β-시트 폴리펩티드 블록 공중합체들(하기 1 내지 4 펩티드)의 구성은 다음과 같다. 본 블록 공중합체들은 하기 도 13에 나타나 있는 고리화 과정 반응을 통하여 N-terminal 부분과 C-terminal 부분이 결합된 거대고리형(macrocyclic) 펩타이드로서, 그 구조는 하기 도 7(청색이라고 표시된 부분은 생체활성 펩티드이고, 적색이라고 표시된 부분은 β-시트 자기조립 부분이다)에 나타내었다.(1) Synthesis Peptides were synthesized using Tribute ™ Peptide Synthesizer (Protein Technologies, Inc) according to standard Fmoc protocol on Rink Amide MBHA Resin LL (Novabiochem), except for Cysteine with methoxytrytil (Mmt) protecting group during synthesis. Standard amino acid protecting groups were used for the remaining amino acids. Synthesis of the synthesized β-sheet polypeptide block copolymers (following 1 to 4 peptide) is as follows. The block copolymers are macrocyclic peptides in which an N-terminal portion and a C-terminal portion are bonded through a cyclization reaction shown in FIG. 13, and the structure thereof is shown in FIG. 7 (blue). Bioactive peptide, and the part marked red is the β-sheet self-assembled part).
1. cyclo[-Phe-Lys-Phe-Glu-Phe-Lys-Phe-Glu-Phe-PEG5-Thr-Arg-Gln-Ala-Arg-Arg-Asn-Arg-Arg-Arg-Arg-Trp-Arg-Arg-PEG5-Cys-] 1.cyclo [-Phe-Lys-Phe-Glu-Phe-Lys-Phe-Glu-Phe-PEG5-Thr-Arg-Gln-Ala-Arg-Arg-Asn-Arg-Arg-Arg-Arg-Trp-Arg -Arg-PEG5-Cys-]
또는 cyclo[-FKFEFKFEF-PEG5-TRQARRNRRRRWRR-PEG5-C-]Or cyclo [-FKFEFKFEF-PEG5-TRQARRNRRRRWRR-PEG5-C-]
2. cyclo[-Trp-Lys-Trp-Glu-Trp-Lys-Trp-Glu-Trp-Lys-Trp-Glu-Trp-Ebes-Gly-Thr-Arg-Gln-Ala-Arg-Arg-Asn-Arg-Arg-Arg-Arg-Trp-Arg-Arg-Ebes-Cys-] 2.cyclo [-Trp-Lys-Trp-Glu-Trp-Lys-Trp-Glu-Trp-Lys-Trp-Glu-Trp-Ebes-Gly-Thr-Arg-Gln-Ala-Arg-Arg-Asn-Arg -Arg-Arg-Arg-Trp-Arg-Arg-Ebes-Cys-]
또는 cyclo[-WKWEWKWEWKWEW-Ebes-GTRQARRNRRRRWRR-Ebes-C-]Or cyclo [-WKWEWKWEWKWEW-Ebes-GTRQARRNRRRRWRR-Ebes-C-]
3. cyclo[-Trp-Trp-Gly-Trp-Trp-Ebes-Thr-Arg-Gln-Ala-Arg-Arg-Asn-Arg-Arg-Arg-Arg-Trp-Arg-Arg-Ebes-] 3.cyclo [-Trp-Trp-Gly-Trp-Trp-Ebes-Thr-Arg-Gln-Ala-Arg-Arg-Asn-Arg-Arg-Arg-Arg-Trp-Arg-Arg-Ebes-]
또는 cyclo[-WWGWW-Ebes-TRQARRNRRRRWRR-Ebes-]Or cyclo [-WWGWW-Ebes-TRQARRNRRRRWRR-Ebes-]
4. cyclo[-Gly-Trp-Trp-Trp-Trp-Ebes-Thr-Arg-Gln-Ala-Arg-Arg-Asn-Arg-Arg-Arg-Arg-Trp-Arg-Arg-Ebes-] 4.cyclo [-Gly-Trp-Trp-Trp-Trp-Ebes-Thr-Arg-Gln-Ala-Arg-Arg-Asn-Arg-Arg-Arg-Arg-Trp-Arg-Arg-Ebes-]
또는 cyclo[-GWWWW-Ebes-TRQARRNRRRRWRR-Ebes-]Or cyclo [-GWWWW-Ebes-TRQARRNRRRRWRR-Ebes-]
(2) 상기 합성된 β-시트 폴리펩티드 블록 공중합체들을 고리화시키기 위해 먼저, 브로모아세트산을 레진이 바인딩된 펩티드의 N-말단에 커플링시키고, 브로모아세트산(28 mg, 200 mmol)과 DIPC(N,N -diisopropylcarbodiimide) 혼합물을 10분 동안 인큐베이션하여 카르복실화시킨 다음에 레진에 첨가하였다. 이후, 레진은 NMP와 DCM으로 세척하였으며, 시스테인으로부터 Mmt기를 제거하기 위하여 레진에 DCM에 용해된 1% TFA를 1분 동안 수차례 가하였다. 이후, 1% DIPEA 3 mL를 가한 후 상온에서 밤새 반응시켜 분자내에서 고리화시켰다. 이어, 레진을 NMP와 아세토니트릴로 세척하고 진공 하에서 건조하였다. 건조된 레진을 분리용액(TFA:TIS:물 = 95:2.5:2.5)으로 3 시간 동안 처리한 다음, t-부틸 메틸 에테르를 이용하여 분말화하였다. 얻어진 펩티드를 역상 HPLC(물-아세토니트릴, 0.1% TFA)로 정제하였다.(2) To cyclize the synthesized β-sheet polypeptide block copolymers, first bromoacetic acid was coupled to the N-terminus of the resin-bound peptide, bromoacetic acid (28 mg, 200 mmol) and DIPC The ( N , N- diisopropylcarbodiimide) mixture was incubated for 10 minutes to carboxylate and then added to the resin. The resin was then washed with NMP and DCM, and 1% TFA dissolved in DCM was added to the resin several times for 1 minute to remove Mmt groups from cysteine. Thereafter, 3 mL of 1% DIPEA was added, followed by reaction at room temperature overnight to cyclize in the molecule. The resin was then washed with NMP and acetonitrile and dried under vacuum. The dried resin was treated with separation solution (TFA: TIS: water = 95: 2.5: 2.5) for 3 hours and then powdered with t-butyl methyl ether. The resulting peptide was purified by reverse phase HPLC (water-acetonitrile, 0.1% TFA).
MALDI-TOF 질량분석을 통해 분자량을 측정하였다. HPLC 분석 결과 펩티드의 순도는 95% 이상 이었다.The molecular weight was determined by MALDI-TOF mass spectrometry. HPLC analysis showed that the purity of the peptide was 95% or more.
물:아세토니트릴 (1:1) 내에서 상기 1번 펩티드는 트립토판의 몰흡광계수를 이용하여 280 nm에서, 그리고 2번 펩티드는 페닐알라닌의 몰흡광계수(195 m-1cm-1)를 이용하여 257.5 nm에서 농도를 분광학적으로 측정하였다.Peptide No. 1 in water: acetonitrile (1: 1) using the tryptophan molar extinction coefficient at 280 nm, and peptide No. 2 using the phenylalanine molar absorption coefficient (195 m −1 cm −1 ). The concentration was measured spectroscopically at 257.5 nm.
실험예 : 고리형(cyclic) β-시트 폴리펩티드 블록Experimental Example: Cyclic β-Sheet Polypeptide Block
(1) 투과전자현미경 분석(TEM)(1) Transmission Electron Microscopy (TEM)
2 mL의 샘플을 탄소 코팅한 구리격자 상에서 완전히 건조시켰다. 이어, 2 mL의 물을 1 분간 가하여 결합되지 않은 펩티드를 용해시키고 여과지를 이용하여 제거하였다. JEOL-JEM 2010 기기를 사용하여 150 kV에서 샘플을 관찰하였다. 얻어진 데이터를 Digital Micrograph™ 소프트웨어를 이용하여 분석하였다.2 mL of sample was completely dried over a carbon coated copper grid. 2 mL of water was then added for 1 min to dissolve the unbound peptide and removed using filter paper. Samples were observed at 150 kV using a JEOL-JEM 2010 instrument. The data obtained were analyzed using Digital Micrograph ™ software.
(2) 원편광 이색성(CD)(2) circularly polarized dichroism (CD)
펠티에 온도조절장치(Applied Photophysics., Ltd)가 구비된 Chirascan™ 원편광 이색성 분광계를 사용하여 CD 스펙트럼을 측정하였다. 스펙트럼은 2 mm 경로길이의 큐벳을 사용하여 260 nm에서부터 200 nm까지 측정하였다. 5회 반복 측정하여 평균값을 기록하였다. 각 아미노산 잔기에 대하여 몰 타원율을 계산하였다. 측정에 앞서 SWCNT가 포함된 또는 포함되지 않은 50 μM 펩티드 용액을 150 mM KF로 실온에서, 또 SWCNT가 포함되지 않은 또다른 50 μM 펩티드 용액을 증류수에서 각각 24 시간 동안 처리하였다.CD spectra were measured using a Chirascan ™ circular dichroism spectrometer equipped with a Peltier thermostat (Applied Photophysics., Ltd). Spectra were measured from 260 nm to 200 nm using cuvettes of 2 mm path length. The average value was recorded by measuring 5 times repeatedly. The mole ellipticity was calculated for each amino acid residue. Prior to the measurement, 50 μM peptide solution with or without SWCNTs was treated with 150 mM KF at room temperature and another 50 μM peptide solution without SWCNTs in distilled water for 24 hours each.
실험예 6.Experimental Example 6.
먼저, 상기 합성예 2의 1번 펩티드를 이용해서 고리형(cyclic)의 폴리펩티드 블록 공중합체도 선형(linear) 폴리펩티드 블록 공중합체와 마찬가지로 SWCNT에 결합하여 그것을 가용화할 수 있는지 확인하였다.First, using the peptide No. 1 of Synthesis Example 2, it was confirmed whether the cyclic polypeptide block copolymer could bind to and solubilize SWCNT like the linear polypeptide block copolymer.
아크방전법을 통해 제조한 SWCNT를 테트라하이드로퓨란(THF)에 현탁하고 동량(5 mg)의 SWCNT를 마이크로 원심분리튜브에 가하였다. THF가 증발한 후, 상기 합성예 2의 1번 고리형 폴리펩티드 블록 공중합체 용액(0.3 mL)을 가하고, 그 혼합물을 실온에서 15 분 동안 초음파 처리하였다.SWCNT prepared by the arc discharge method was suspended in tetrahydrofuran (THF) and the same amount (5 mg) of SWCNT was added to the microcentrifuge tube. After THF evaporated, the solution of No. 1 cyclic polypeptide block copolymer of Synthesis Example 2 (0.3 mL) was added, and the mixture was sonicated at room temperature for 15 minutes.
하기 도 8(좌측 이미지)에서 보이는 바와 같이 낮은 농도(20 mM)의 염 수용액에 포함된 1번 펩티드는 SWCNT를 효과적으로 가용화시키지 못한 반면, 150 mM의 염 농도 수용액에서 1번 펩티드는 초음파 처리의 도움을 받아 도 8(우측 이미지)과 같이 대부분의 SWCNT를 현탁시켰다. 이러한 결과로부터 고리형 폴리펩티드 공중합체 역시 선형 폴리펩티드 공중합체와 마찬가지로 적절한 이온세기에서 페닐알라닌 잔기의 소수성 및 π-π 스태킹 상호작용과 생체활성 부분의 수용액과의 상호작용을 통해 펩티드/SWCNT 복합체의 용해성을 향상시킬 수 있음을 알 수 있다.As shown in FIG. 8 (left image), peptide 1 contained in a low concentration (20 mM) salt solution did not effectively solubilize SWCNT, whereas peptide 1 in a 150 mM salt concentration solution aided in sonication. Received most of the SWCNT as shown in Figure 8 (right image). From these results, cyclic polypeptide copolymers, like linear polypeptide copolymers, enhance the solubility of peptide / SWCNT complexes through hydrophobicity and π-π stacking interactions of phenylalanine residues and aqueous solutions of bioactive moieties at appropriate ionic strengths. It can be seen that.
실험예 7.Experimental Example 7.
하기 도 9에서 보이는 바와 같이 TEM 이미지로부터 고리형 폴리펩티드 공중합체가 실제로 SWCNT를 가용화시킨 모습을 더욱 분명하게 확인할 수 있다.As shown in FIG. 9, it can be more clearly seen that the cyclic polypeptide copolymer actually solubilizes the SWCNT from the TEM image.
실험예 8.Experimental Example 8.
제조된 펩티드/SWCNT 복합체의 펩티드 α-나선구조 안정화 기능을 확인하기 위해서 CD 스펙트럼을 측정하였다.CD spectra were measured to confirm peptide α-helix stabilization function of the prepared peptide / SWCNT complex.
α-나선구조를 지탱하는 단백질 모방(mimic)으로서 SWCNT의 기능을 검증하기 위해 50 μM 농도의 상기 합성예 2의 2번 펩티드를 순수한 물과 150 mM의 염 수용액에 녹여서 각각 CD 스펙트럼을 측정하였고(도 10의 b(점선), c(이점쇄선) 스펙트럼), 또 다른 50 μM 농도의 2번 펩티드를 150 mM 염 수용액에 포함되어있는 20 ㎍ SWCNT에 가한 후 초음파 처리를 통해 펩티드/SWCNT 복합체를 형성하여 CD 스펙트럼 측정에 이용하였다(도 10의 a(실선) 스펙트럼).In order to verify the function of SWCNT as a protein mimic supporting the α-helix structure, peptide 2 of Synthesis Example 2 at a concentration of 50 μM was dissolved in pure water and an aqueous solution of 150 mM salt, and the CD spectra were measured. 10 (b), c (double dashed line) spectrum of Figure 10, another 50 μM peptide No. 2 was added to 20 μg SWCNT contained in 150 mM aqueous solution and then sonicated to form a peptide / SWCNT complex Was used for the CD spectrum measurement (a (solid line) spectrum in Fig. 10).
하기 도 10에서 확인할 수 있듯이 SWCNT가 포함되지 않은 펩티드는 물과 염 수용액 상에서 α-나선구조를 안정화시키는 모습을 보여주지 못했지만, 염 수용액 내의 펩티드/SWCNT 복합체의 CD 스펙트럼은 208 nm와 222 nm에서 눈에 띄는 타원율의 음의 최대값을 나타냄으로써 α-나선구조를 효과적으로 안정화시키고 있음을 밝히고 있다. 이러한 결과로부터 SWCNT는 소수성 및 π-π 스태킹 상호작용을 통해 고리형 폴리펩티드 공중합체의 β-시트 형성 부분을 튼튼하게 지탱함으로써 α-나선구조를 지탱하는 체내의 거대 단백질과 유사하게 펩티드의 α-나선구조를 안정화시키고 있음을 확인할 수 있다.As can be seen in FIG. 10, the peptide without SWCNT did not show stabilization of α-helical structure in water and salt solution, but the CD spectrum of peptide / SWCNT complex in salt solution was observed at 208 nm and 222 nm. By showing a negative maximum value of the ellipticity, it is found that the α-helical structure is effectively stabilized. From these results, SWCNTs strongly support the β-sheet-forming part of the cyclic polypeptide copolymer through hydrophobic and π-π stacking interactions, similar to the macromolecules in the body that support the α-helix structure. It can be seen that the structure is stabilized.
실험예 9.Experimental Example 9.
SWCNT의 가용화를 위한 폴리펩티드 공중합체의 β-시트 형성 부분으로서 페닐알라닌 잔기 외에 트립토판 잔기의 이용 가능성을 모색하기 위해 2번 고리형 펩티드를 SWCNT 가용화에 적용시켜 보았다. Cyclic peptide 2 was applied to SWCNT solubilization to explore the availability of tryptophan residues in addition to phenylalanine residues as β-sheet forming parts of the polypeptide copolymers for solubilization of SWCNTs.
도 11(좌측 이미지)에서 보이는 바와 같이 펩티드가 포함되지 않은 염농도 150 mM의 물은 SWCNT를 효과적으로 가용화시키지 못한 반면 10 μM 농도의 1번 펩티드가 150 mM의 KF 염 용액에 포함된 도 11(우측 이미지)의 샘플은 초음파 처리의 도움을 받아 대부분의 SWCNT를 현탁시켰다. 이러한 결과로부터, 트립토판 잔기는 페닐알라닌 잔기와 마찬가지로 소수성 및 π-π 스태킹 상호작용을 통해 SWCNT를 효과적으로 가용화 시킬 수 있음을 알 수 있다.As shown in FIG. 11 (left image), salt concentration of 150 mM without peptide did not effectively solubilize SWCNT, whereas peptide No. 1 at 10 μM concentration was contained in 150 mM KF salt solution (right image). The samples in) suspended most of the SWCNTs with the aid of sonication. From these results, it can be seen that tryptophan residues, like phenylalanine residues, can effectively solubilize SWCNTs through hydrophobic and π-π stacking interactions.
실험예 10.Experimental Example 10.
β-시트 폴리펩티드 부분의 β-시트 형성 잔기, 특히 트립토판 잔기의 개수와 배치를 달리하여도 SWCNT를 가용화시킬 수 있음을 확인하기 위해 상기 합성예 2의 3번, 4번 고리형 펩티드를 적용시켜 보았다.In order to confirm that SWCNTs can be solubilized by varying the number and arrangement of β-sheet forming residues, especially tryptophan residues of the β-sheet polypeptide moiety, the 3 and 4 cyclic peptides of Synthesis Example 2 were applied. .
도 12와 같이 40 mM의 KF 염 수용액은 SWCNT를 잘 현탁시키지 못한 반면(좌측 이미지) 40 mM 염 수용액에 포함된 30 μM 농도의 3번 펩티드는 초음파 처리의 도움을 받아 SWCNT를 가용화 시키는 경향성을 나타내는 것을 확인할 수 있었다(우측 이미지).As shown in FIG. 12, the 40 mM aqueous KF salt solution did not suspend the SWCNT well (left image) while the peptide of 30 μM concentration contained in the 40 mM aqueous salt solution showed a tendency to solubilize the SWCNT with the help of sonication. It was confirmed (right image).
이러한 결과에 비춰볼 때, 또 상기 실험예들의 결과를 종합적으로 생각해 볼 때 β-시트 폴리펩티드 부분의 잔기들에 대한 종류와 수량, 배치를 적절히 달리하여도 SWCNT 가용화에 적용시킬 수 있음을 확인할 수 있다. In view of these results, and comprehensively considering the results of the above experimental examples, it can be confirmed that the SWCNT solubilization can be applied even if the kind, quantity, and arrangement of the residues of the β-sheet polypeptide moiety are appropriately changed.
이와 같이, 생체활성 β-시트 폴리펩티드 블록 공중합체와 CNT의 조합 자기조립 과정에서 여러 개의 상이한 모드가 관련됨을 확인하였다. 생체활성 β-시트 폴리펩티드 블록 공중합체는 스스로 자기조립할 수 있으며, 경우에 따라서는 CNT-복합체의 기능화에도 이용될 수 있다. 이것은 생체활성 β-시트 폴리펩티드 블록 공중합체의 응용범위를 넓히는 데 있어 중요한 의미를 갖는다. 생체활성 β-시트 폴리펩티드 블록 공중합체/CNT 복합체는 자극 반응성 바이오 소재로서 또는 CNT 기반 전자 바이오 센서 장치의 제작에 사용될 수 있다. 또한, β-시트 펩티드와 탄소 기반 소수성 물질 사이의 상호작용에 대한 이해는 단백질의 이상접힘에 의한 질환에 대한 억제제의 설계 및 개발에도 유용할 것으로 기대된다.As such, it was found that several different modes are involved in the combinatorial self-assembly process of the bioactive β-sheet polypeptide block copolymer and CNT. Bioactive β-sheet polypeptide block copolymers can self-assemble themselves and, if desired, also be used for functionalization of CNT-complexes. This is important for broadening the application of bioactive β-sheet polypeptide block copolymers. Bioactive β-sheet polypeptide block copolymers / CNT complexes can be used as stimuli reactive biomaterials or in the fabrication of CNT based electronic biosensor devices. In addition, understanding of the interaction between β-sheet peptides and carbon-based hydrophobic materials is expected to be useful in the design and development of inhibitors for diseases caused by abnormal folding of proteins.

Claims (12)

  1. β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체로서,As a complex of β-sheet polypeptide block copolymer and carbon nanotube,
    상기 β-시트 폴리펩티드 블록 공중합체는 β-시트 폴리펩티드 블록 및 생체활성 폴리펩티드 블록으로 이루어지고,The β-sheet polypeptide block copolymer consists of a β-sheet polypeptide block and a bioactive polypeptide block,
    상기 β-시트 폴리펩티드 블록은 비극성 아미노산과 극성 아미노산이 교대로 반복되는 구조, 또는 비극성 아미노산이 50-100%인 구조를 가지며,The β-sheet polypeptide block has a structure in which non-polar amino acids and polar amino acids are alternately repeated, or 50-100% of non-polar amino acids,
    상기 생체활성 폴리펩티드 블록은 생체활성 폴리펩티드 블록을 이루는 아미노산의 50-100%가 극성 아미노산으로 이루어지며,The bioactive polypeptide block is composed of polar amino acids 50-100% of the amino acids constituting the bioactive polypeptide block,
    상기 β-시트 폴리펩티드 블록은 상기 탄소나노튜브 표면과 비공유 결합을 하고,The β-sheet polypeptide block is non-covalently bonded to the surface of the carbon nanotubes,
    상기 생체활성 폴리펩티드 블록은 전하를 띠면서 상기 복합체 외곽으로 노출되는 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체.The bioactive polypeptide block is charged and exposed to the outside of the complex complex with a β-sheet polypeptide block copolymer and carbon nanotubes.
  2. 제 1 항에 있어서,The method of claim 1,
    상기 비극성 아미노산은 페닐알라닌(phenylalanine), 알라닌(alanine), 발린(valine), 이소류신(isoleucine), 류신(leucine), 메치오닌(methionine), 티로신(tyrosine) 및 트립토판(tryptophan) 중에서 선택되고,The non-polar amino acid is selected from phenylalanine (alanine), alanine (alanine), valine (valine), isoleucine (isoleucine), leucine, leucine, methionine, tyrosine (tyrosine) and tryptophan,
    상기 극성 아미노산은 라이신(lysine), 글라이신(glycine), 아르기닌(arginine), 프롤린(proline), 글루타민(glutamine), 세린(serine), 히스티딘(histidine), 아스파라긴산(aspartic acid), 글루타민산(glutamic acid), 트레오닌(threonine), 아스파라긴(aspargine), 시스테인(cysteine) 및 셀레노시스테인(selenocysteine) 중에서 선택되는 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체.The polar amino acids are lysine, glycine, arginine, arginine, proline, glutamine, glutamine, serine, histidine, aspartic acid and glutamic acid. Β-sheet polypeptide block copolymer and a complex with carbon nanotubes, characterized in that selected from threonine (threonine), asparagine (aspargine), cysteine (cysteine) and selenocysteine (selenocysteine).
  3. 제 2 항에 있어서,The method of claim 2,
    상기 β-시트 폴리펩티드 블록의 비극성 아미노산은 페닐알라닌 또는 트립토판이고, 상기 극성 아미노산은 라이신, 글루탐산 또는 글라이신인 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체.The non-polar amino acid of the β-sheet polypeptide block is phenylalanine or tryptophan and the polar amino acid is a lysine, glutamic acid or glycine complex with β-sheet polypeptide block copolymers and complexes with carbon nanotubes.
  4. 제 3 항에 있어서,The method of claim 3, wherein
    상기 β-시트 폴리펩티드 블록은 펩티드 가닥 사이에서 β-시트 수소결합을 형성하여 자기조립을 형성하고, 상기 페닐알라닌 또는 트립토판이 상기 탄소나노튜브 표면과 π-π 스태킹 및 소수성 상호작용을 통하여 비공유 결합을 형성하는 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체.The β-sheet polypeptide block forms β-sheet hydrogen bonds between peptide strands to form self-assembly, and the phenylalanine or tryptophan forms non-covalent bonds through π-π stacking and hydrophobic interactions with the carbon nanotube surface. It is characterized in that the β-sheet polypeptide block copolymer and a complex with carbon nanotubes.
  5. 제 1 항에 있어서,The method of claim 1,
    상기 β-시트 폴리펩티드 블록 공중합체는 N-말단과, C-말단이 결합하여 거대 고리형(macrocyclic) 펩티드인 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체.The β-sheet polypeptide block copolymer is a complex of β-sheet polypeptide block copolymer and carbon nanotubes, characterized in that the N-terminal and the C-terminal are combined to be a macrocyclic peptide.
  6. 제 1 항 내지 제 5 항 중 어느 한 항에 따른 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체, 및A complex of the β-sheet polypeptide block copolymer according to any one of claims 1 to 5 and a carbon nanotube, and
    상기 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체에 표적 바이오 물질과 반응할 수 있는 리간드 또는 리셉터가 부착된 것을 특징으로 하는 바이오 센서.And a ligand or receptor capable of reacting with a target biomaterial to a complex of the β-sheet polypeptide block copolymer and carbon nanotubes.
  7. 제 6 항에 있어서,The method of claim 6,
    상기 리간드 및 리셉터는 각각 독립적으로 효소기질, 리간드, 아미노산, 펩티드, 단백질, 효소, 지질, 코펙터, 탄수화물 및 이들 2종의 조합인 것을 특징으로 하는 바이오 센서.The ligand and the receptor are each independently an enzyme substrate, a ligand, an amino acid, a peptide, a protein, an enzyme, a lipid, a cofactor, a carbohydrate and a combination of these two.
  8. 제 1 항 내지 제 5 항 중 어느 한 항에 따른 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체를 유효 성분으로 포함하는 생물학적 활성 물질의 세포내 전달용 조성물.A composition for intracellular delivery of a biologically active substance comprising a β-sheet polypeptide block copolymer according to any one of claims 1 to 5 and a complex with a carbon nanotube as an active ingredient.
  9. (a) 탄소나노튜브 현탁액에서 현탁 용매를 제거함으로써 전처리된 탄소나노튜브를 수득하는 단계; 및(a) obtaining a pretreated carbon nanotube by removing the suspension solvent from the carbon nanotube suspension; And
    (b) 상기 전처리된 탄소나노튜브를 β-시트 폴리펩티드 블록 공중합체 수용액에 추가하여 분산액을 수득하는 단계;를 포함하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체의 제조방법.(b) adding the pre-treated carbon nanotubes to the aqueous β-sheet polypeptide block copolymer solution to obtain a dispersion; and a method of preparing a complex of the β-sheet polypeptide block copolymer and the carbon nanotubes.
  10. 제 9 항에 있어서,The method of claim 9,
    상기 분산액은 염을 추가로 포함하는 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체의 제조방법.The dispersion is a method for producing a complex of β-sheet polypeptide block copolymers and carbon nanotubes further comprises a salt.
  11. 제 9 항 또는 제 10 항에 있어서,The method according to claim 9 or 10,
    상기 (a) 단계의 상기 현탁 용매는 테트라 하이드로 퓨란이고, 상기 현탁 용매는 원심분리 방법에 의해서 제거되는 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체의 제조방법.The suspension solvent of step (a) is tetrahydrofuran, the suspension solvent is a method for producing a complex of β-sheet polypeptide block copolymers and carbon nanotubes, characterized in that the removal by centrifugation.
  12. 제 10 항에 있어서,The method of claim 10,
    상기 염의 상기 분산액 내 농도가 10-150 mM이고, 상기 분산액은 초음파 방법을 이용하여 제조되는 것을 특징으로 하는 β-시트 폴리펩티드 블록 공중합체 및 탄소나노튜브와의 복합체의 제조방법.The concentration of the salt in the dispersion is 10-150 mM, the dispersion is a method for producing a β-sheet polypeptide block copolymer and a complex with carbon nanotubes, characterized in that prepared by using an ultrasonic method.
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